WO2022239720A1 - Antibody having reduced binding affinity for antigen - Google Patents

Antibody having reduced binding affinity for antigen Download PDF

Info

Publication number
WO2022239720A1
WO2022239720A1 PCT/JP2022/019622 JP2022019622W WO2022239720A1 WO 2022239720 A1 WO2022239720 A1 WO 2022239720A1 JP 2022019622 W JP2022019622 W JP 2022019622W WO 2022239720 A1 WO2022239720 A1 WO 2022239720A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
apd
antibodies
antigen
modified
Prior art date
Application number
PCT/JP2022/019622
Other languages
French (fr)
Japanese (ja)
Inventor
一則 片岡
タオ ヤン
祐希 持田
泰孝 安楽
オラシオ カブラル
宏昭 喜納
Original Assignee
公益財団法人川崎市産業振興財団
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 公益財団法人川崎市産業振興財団 filed Critical 公益財団法人川崎市産業振興財団
Priority to JP2023521007A priority Critical patent/JPWO2022239720A1/ja
Priority to EP22807422.5A priority patent/EP4342497A1/en
Publication of WO2022239720A1 publication Critical patent/WO2022239720A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule

Definitions

  • the present invention relates to antibodies with reduced binding affinity to antigens.
  • Antibody drugs are being developed for the treatment of diseases around the world. Antibodies are molecularly targeted drugs and are therefore ideally specific to the target tissue. In practice, however, antibody antigens are expressed in tissues other than target tissues, and therefore are not completely target tissue-specific in many cases. In particular, anticancer agents containing antibodies targeting cancer antigens may damage areas other than tumors and cause side effects if the target tissue specificity is low.
  • Glioblastoma the most aggressive brain tumor, is characterized by the highest mortality, short survival, and poor prognosis, and is universally fatal (Non-Patent Document 1- 3).
  • Immune checkpoint inhibitor (ICB) therapy using monoclonal antibodies has revolutionized cancer therapy (Non-Patent Documents 4-6).
  • BBB blood-brain barrier
  • glioblastoma GBM
  • ICB therapy may cause immune-related adverse events (irAE) such as autoimmune activation, lymphocyte infiltration, and release of inflammatory cytokines (Non-Patent Documents 10-13).
  • Non-Patent Documents 14-16 In ICB treatment, PD-L1 is expressed in vascular endothelial cells, muscle, hepatocytes, and pancreatic islet cells, so that off-target binding to normal tissues always occurs, thus anti-PD-L1 antibodies (aPD- When L1) is administered systemically, the incidence of irAEs increases and the therapeutic effect decreases (Non-Patent Documents 14-16).
  • antibodies with reduced binding affinity to antigens are provided.
  • the present invention also provides antibodies that reactivate in environmental responsiveness.
  • the present inventors have found that by modifying an antibody with a non-charged hydrophilic polymer and covering the antibody to reduce the affinity of the antibody for the antigen, the antibody is inactivated in non-target tissues in the body (to the antigen). with reduced or lost binding affinity), and the linker is designed to cleave the modification with the uncharged hydrophilic polymer within the target tissue, removing the modification from the antibody within the target tissue.
  • the inventors have found that by allowing the antibody to be activated, it is possible to activate the antibody (a state in which the affinity for the antigen is higher) only in the target tissue in the body.
  • a modified antibody modified with an uncharged hydrophilic polymer block preferably polyethylene glycol (PEG)
  • PEG polyethylene glycol
  • each linkage may be via a spacer
  • the environmentally responsive bond is a bond that cleaves under a reducing environment
  • the modified antibody ⁇ here, although not particularly limited
  • the antibody is preferably an immune checkpoint molecule binds to and inhibits immune checkpoints ⁇ .
  • [3] The antibody of [1] above, whose binding affinity (KD) for the antigen is 5% or less compared to the unmodified antibody (unmodified antibody).
  • the antibody of [1] above which does not substantially or significantly bind to its antigen in a serum environment ⁇ eg, binding is below the detection limit ⁇ .
  • the antibody of [5] above, wherein the binding affinity (KD) for the antigen is restored when the environment-responsive bond is cleaved.
  • the GLUT1 ligand is glucose.
  • the antibody of [15] above, wherein the immune checkpoint molecule is a counterpart of an immune checkpoint molecule expressed on immune cells.
  • a pharmaceutical composition comprising the antibody of any one of [1] to [17] above.
  • a pharmaceutical composition comprising the antibody of [18] above.
  • the antibody of [21] above, wherein the tumor antigen is a non-brain tumor antigen.
  • ADC drug-antibody conjugate
  • the ADC is antibody moiety-(linker-drug) n ⁇ where n is the number average and ranges from 2-10. ⁇ .
  • the ADC has 2 to 4 -linker-drugs introduced to the side chain amino groups of the antibody moiety, and has an average hydrodynamic diameter (volume average) of 12 nm to 30 nm (especially 13 nm to 25 nm), the antibody according to [30] above.
  • the antibody of [35] above, wherein the unmodified ADC has an average hydrodynamic diameter (volume average) of 12 nm or less.
  • the antibody of any of the above, wherein the antigen to which the antibody portion binds is a cancer antigen.
  • a composition comprising the antibody of any of the above.
  • ADC is gemtuzumab ozogamicin (Milotarg), ibritumomab tiuxetan (Zevalin), brentuximab vedotin (ADCETRIS), trastuzumab emtansine (Kadcyra), inotuzumab ozogamicin (Vesponsa), moxetumomab pasudotox-tdfk (LUMOXITI), polatuzumab iq vedotin (Polivy), trastuzumab deruxtecan (Enherts), and enfortumab vedotin (PADCEV).
  • a composition comprising the antibody of [40] above.
  • An antibody that binds to an immune checkpoint molecule and inhibits the immune checkpoint which is modified with an uncharged hydrophilic polymer block (preferably polyethylene glycol (PEG)),
  • An antibody e.g., a modified antibody in which an environmentally responsive bond and an antibody are linked in that order, each linkage may be via a spacer, and the environmentally responsive bond is a bond that is cleaved under a reducing environment .
  • the antibody of [1] above, whose binding affinity (KD) for the antigen is 5% or less compared to before the modification.
  • [4] The antibody of [1] above, which does not substantially or significantly bind to its antigen in a serum environment ⁇ eg, binding is below the detection limit ⁇ .
  • [5] The antibody according to any one of [1] to [4] above, wherein the environment-responsive bond is a bond that cleaves under a reducing environment in brain parenchyma or in a reducing environment in tumor tissue.
  • [6] The antibody of [5] above, wherein the binding affinity (KD) for the antigen is restored when the environment-responsive bond is cleaved.
  • the antibody that binds to the immune checkpoint molecule and inhibits the immune checkpoint is an antibody that binds to the immune checkpoint molecule and neutralizes the interaction between the immune checkpoint molecules [1] to The antibody according to any one of [14].
  • the antibody of the present invention and the in vivo behavior of the antibody are shown.
  • the antibody and the uncharged hydrophilic polymer block are linked via a reducing environment-responsive linker.
  • Antibodies have reduced or abolished binding affinity for antigens due to linkage with uncharged hydrophilic polymer blocks.
  • the antibody is inactivated outside the target organ (including non-target tissue).
  • FIG. 1 an example of antibody delivery to and function in the brain is shown.
  • the reducing-environment-responsive linker is cleaved in response to the reducing environment within the brain parenchyma.
  • antibodies can be reactivated and exert their action against their antigens only in the brain, in tumor tissue, and other special circumstances where disulfide bonds are cleaved.
  • An anti-PD-L1 antibody and a non-charged hydrophilic polymer block (eg, PEG) with a targeting molecule at one end are linked via a reduced environment-responsive linker.
  • Modified antibodies of the invention with reduced binding affinity show a reduction in size due to the removal of the modification sites from the antibody in a reducing environment, reverting to approximately the size of the unmodified antibody (left panel).
  • the right panel shows that antibodies are modified with an uncharged hydrophilic polymer block to lose zeta potential and are released from the uncharged hydrophilic polymer block in a reducing environment to restore zeta potential.
  • Figure 2 shows the structure of a linker in a preferred embodiment of the present invention and, when used, cleavage of the linker in a reducing environment yields or releases a native antibody (unmodified antibody). It shows that one embodiment of the antibody of the present invention restores its binding affinity to its antigen in a reducing environment-dependent manner.
  • Gluc-S-aPD-L1 has virtually lost its binding affinity for antigen (see middle bar), but under reducing conditions (Gluc-S-aPD-L1/GSH) its binding affinity recovered to the level of unmodified antibody.
  • FIG. 4 is a graph showing cell staining images (upper panel) and the amount of binding (fluorescence intensity) showing that an antibody whose binding is restored in a reducing environment recognizes PD-L1 on the cell surface. Retention of antibody in blood (bottom left panel) and amount of antibody in plasma (bottom right panel) are shown. The unmodified anti-PD-L1 antibody showed binding to the inner wall of blood vessels, whereas the modified antibody showed no substantial binding. Antibodies modified with uncharged hydrophilic polymer blocks were obtained with 0%, 25%, 50%, 100% of the uncharged hydrophilic polymer blocks having glucose at one end.
  • FIG. 9 shows the accumulation of various antibodies in brain tumors.
  • FIG. 1 shows a schematic diagram of a system for detecting an antibody of one embodiment of the present invention with a secondary antibody.
  • the antibody is modified with an uncharged hydrophilic polymer block to prevent access and recognition of the antibody by the secondary antibody.
  • antibodies released from uncharged hydrophilic polymer blocks are recognized by secondary antibodies.
  • modifications are removed from the antibody of one aspect of the invention to show that the antibody of the invention is recognized by a secondary antibody.
  • Flow cytometry results showing increased cytotoxic T cells in the brains of mice treated with various antibodies.
  • Immunochemical histological staining results showing increased cytotoxic T cells in the brains of mice treated with various antibodies.
  • FIG. 1 shows the concentration of interferon gamma (IFN ⁇ ) in blood of mice after stimulation of antigen processing and presentation mechanisms in mice administered with various antibodies.
  • IFN ⁇ interferon gamma
  • the abundance of immunosuppressive Foxp3+ T cells in mice administered with various antibodies is shown.
  • the abundance of CD44hiCD62Llow effector memory T cell subsets in the spleen of mice treated with various antibodies is shown.
  • a scheme is shown in which an antibody of one embodiment of the present invention is administered to a mouse grafted with a tumor in the right brain, then a tumor is grafted in the left brain, and the tumor is observed.
  • the presence of tumors in the left brain (left panel) and Kaplan-Meier curves (right panel) are shown.
  • CD45+ cell infiltration in non-target tissues (lung, kidney and liver) of mice after administration of various antibodies is shown.
  • TNF- ⁇ , IL-6 and IL-1 ⁇ levels in non-target tissues (lung, kidney and liver) of mice after administration of various antibodies are shown.
  • the antibody of one embodiment of the present invention cannot be recognized by a secondary antibody in non-target tissues (lung, kidney, and liver) of mice after administration of various antibodies, that is, the modified site is not removed from the antibody in non-target tissues. Indicates an inactive state.
  • Schematic representation of a modified antibody with no targeting molecule consisting of an uncharged hydrophilic polymer block (eg, PEG), a reducing environment-responsive polymer, and an antibody linked in that order.
  • FIG. 21 shows the anti-tumor effect when a modified antibody having the structure shown in FIG.
  • Fig. 2 shows temporal changes in the particle size distribution of a PEG-modified antibody under a reducing environment simulating the reducing environment in cancer tissue in vivo and changes in the intensity of scattered light.
  • a "subject" is a mammal including a human.
  • a subject may be a healthy subject or a subject suffering from any disease.
  • treatment includes both therapeutic treatment and prophylactic treatment.
  • treatment means treating, curing, preventing or ameliorating the remission of a disease or disorder, or reducing the rate of progression of a disease or disorder.
  • prevention means reducing the likelihood of developing a disease or condition or delaying the onset of a disease or condition.
  • disease means a condition for which treatment is beneficial.
  • cancer generally means highly malignant tumors such as malignant tumors and brain tumors. Tumors include benign and malignant tumors.
  • blood-brain barrier refers to a functional barrier that exists between the blood (or blood circulation) and the brain and has selectivity for the permeation of substances. It is believed that the blood-brain barrier is actually composed of cerebrovascular endothelial cells and the like. Although there are many unclear points about the substance permeability of the blood-brain barrier, it is known that glucose, alcohol and oxygen easily pass through the blood-brain barrier, and fat-soluble substances and small molecules (for example, molecular weight less than 500) It is believed that they tend to pass through more easily than water-soluble molecules or macromolecules (eg, molecular weight of 500 or more).
  • blood-nerve barrier refers to a functional barrier that exists between the blood circulation and peripheral nerves and has selectivity for the permeation of substances.
  • blood-cerebrospinal fluid barrier refers to a functional barrier that exists between the blood circulation and the cerebrospinal fluid and is selective for the permeation of substances.
  • blood-retinal barrier refers to a functional barrier that exists between the blood circulation and retinal tissue and is selective for the permeation of substances.
  • the blood-nerve barrier, blood-cerebrospinal fluid barrier, and blood-retinal barrier are believed to be vascular endothelial cells and the like present in each barrier, and their functions are believed to be similar to those of the blood-brain barrier.
  • targeting molecule refers to a target molecule (e.g., an antigen such as a protein on the cell surface) present on the surface of a cell in vivo. It is the molecule that binds. Since the targeting molecule has binding affinity for the target molecule, it can have the effect of actively delivering (or concentrating) the substance linked to the targeting molecule to the location where the target molecule exists.
  • Targeting molecules can be molecules (eg, antibodies, antigen-binding fragments thereof, aptamers, peptides, lectins, etc.) that have binding affinity for target molecules expressed on cell surfaces (eg, endothelial cell surfaces).
  • Antibodies without targeting molecules or modified with uncharged hydrophilic polymer blocks are passively delivered to tissues (eg, tumor tissues) by, for example, the EPR effect.
  • An antibody modified with an uncharged hydrophilic polymer block bearing a targeting molecule can bind to the target molecule in an in vivo environment.
  • a "targeting molecule" may be linked to the modified antibody portion of the invention (preferably attached to the distal end of the uncharged hydrophilic polymer block; where the proximal end is the side that is linked to the antibody, and the distal end is the side that is not linked to the antibody), which is a molecular moiety separate from the antibody moiety.
  • GLUT1 ligand means a substance that specifically binds to GLUT1.
  • Various ligands are known as GLUT1 ligands, including but not limited to molecules such as glucose and hexose, any of which can be used in the preparation of antibodies modified with uncharged hydrophilic polymer blocks in the present invention. can be used.
  • GLUT1 ligands preferably have an affinity for GLUT1 that is equal to or greater than that of glucose.
  • GLUT1 ligands include GLUT1-binding molecules, and GLUT1-binding molecules include GLUT1-binding aptamers.
  • GLUT1 ligands with higher specificity to GLUT1 can be preferably used.
  • the GLUT1 ligand can be glucose.
  • antibody means an immunoglobulin, which has a structure in which two heavy chains (H chains) and two light chains (L chains) stabilized by a pair of disulfide bonds are associated.
  • the heavy chain consists of a heavy chain variable region VH, heavy chain constant regions CH1, CH2, CH3, and a hinge region located between CH1 and CH2, and the light chain consists of a light chain variable region VL and a light chain constant region CL.
  • a variable region fragment (Fv) consisting of VH and VL is a region that directly participates in antigen binding and imparts diversity to antibodies.
  • the antigen-binding region consisting of VL, CL, VH and CH1 is called the Fab region, and the region consisting of the hinge region, CH2 and CH3 is called the Fc region.
  • the regions that directly contact the antigen undergo particularly large changes and are called complementarity-determining regions (CDRs).
  • CDRs complementarity-determining regions
  • a portion other than the CDRs with relatively few mutations is called a framework region (FR).
  • the light chain and heavy chain variable regions each have three CDRs, which are referred to as heavy chain CDRs 1-3 and light chain CDRs 1-3 in order from the N-terminus.
  • modified antibody means an antibody that has a chemical modification. Chemical modifications include modification with uncharged hydrophilic polymers (eg, polyethylene glycol, polyoxazolines).
  • an antibody may be a monoclonal antibody or a polyclonal antibody.
  • an antibody can be of any isotype, IgG, IgM, IgA, IgD, IgE. It may be prepared by immunizing non-human animals such as mice, rats, hamsters, guinea pigs, rabbits, chickens, etc., or may be recombinant antibodies, chimeric antibodies, humanized antibodies, fully humanized antibodies. etc.
  • a chimeric antibody refers to an antibody in which antibody fragments derived from different species are linked.
  • Humanized antibody means an antibody in which the corresponding positions of a human antibody are substituted with an amino acid sequence characteristic of a non-human antibody, for example, the heavy chain of an antibody produced by immunizing a mouse or rat Those having CDRs 1-3 and light chain CDRs 1-3, with all other regions derived from human antibodies, including the four framework regions (FRs) each of the heavy and light chains. Such antibodies are sometimes referred to as CDR-grafted antibodies.
  • the term “humanized antibody” may also include human chimeric antibodies.
  • a “human chimeric antibody” is a non-human antibody in which the constant region of the non-human antibody is replaced with the constant region of a human antibody.
  • Antibodies can be isolated. The antibody is preferably an isolated monoclonal antibody for pharmaceutical products. Antibodies may have antibody dependent cellular cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC).
  • the term "antigen-binding fragment of an antibody” refers to a fragment of an antibody that maintains antigen-binding ability.
  • Fab' consisting of VL, VH, CL and CH1 regions and Fab' having a hinge region; F(ab')2 in which two Fabs are linked by a disulfide bond at the hinge region; consisting of VL and VH Fv;
  • scFv which is a single-chain antibody in which VL and VH are linked by an artificial polypeptide linker
  • multi- (or bi)specific antibodies such as diabodies, scDb, tandem scFv, and leucine zippers. include, but are not limited to.
  • CDRs are complementarity determining regions present in the heavy and light chain variable regions of antibodies. There are three each in the heavy and light chain variable regions, designated from the N-terminus as CDR1, CDR2 and CDR3. CDRs are, for example, numbered by Kabat et al. can be determined based on
  • antigen refers to a substance that an antibody can bind to.
  • Antigens can be immunogenic.
  • Antigens can be proteins, nucleic acids, metabolites, and the like.
  • alkyl means straight (ie, unbranched) or branched carbon chains, or combinations thereof.
  • alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl, and these isomers are included.
  • alkyl can be C1 alkyl, C2 alkyl , C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, or C8 alkyl .
  • alkenyl refers to a group having a double bond between two adjacent carbons of alkyl.
  • alkynyl refers to the group having triple bonds in two adjacent carbocyclic rings of alkyl.
  • lower means having 1 to 8 carbon atoms, such as 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, It can mean 2-5, 2-4, or 2-3.
  • optionally substituted means that a compound or group may or may not be substituted (i.e., substituted or unsubstituted) by other groups. do. Said other groups include halogens, hydroxyl groups, lower alkyl groups, and preferably negatively charged groups such as carboxyl groups.
  • hypoglycemia or lowering blood glucose levels refers to lowering blood glucose levels in a subject below what they would have had without the treatment.
  • Hypoglycemia can be, for example, a level of hypoglycemia (eg, 70 mg/dL) or higher that does not cause autonomic symptoms such as fatigue, hand tremors, palpitations, tachycardia, or cold sweats.
  • Hypoglycemia is a level that does not cause central nervous system symptoms (e.g., strong weakness, fatigue, blurred vision, headache, drowsiness, etc.) and cerebral dysfunction (e.g., decreased level of consciousness, abnormal behavior, convulsions, coma, etc.).
  • the blood sugar level can be lowered, for example, to about 80-100 mg/dL.
  • Inducing hypoglycemia is used in the sense of including producing fasting blood sugar.
  • Hypoglycemia can also be induced, for example, by fasting.
  • Methods for controlling hypoglycemia include administration of antidiabetic drugs. For example, when controlling to a hypoglycemic state, it is permissible, for example, to take other drugs or drink beverages such as water as long as the goal of controlling to a hypoglycemic state is achieved. Inducing hypoglycemia may be accompanied by other treatments that do not substantially affect blood glucose.
  • fasting refers to subjecting a subject to fasting, e.g. 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, 19 hours or more, 20 hours or more, 21 hours or more, 22 hours or more, 23 hours 24 hours or more, 25 hours or more, 26 hours or more, 27 hours or more, 28 hours or more, 29 hours or more, 30 hours or more, 31 hours or more, 32 hours or more, 33 hours or more, 34 hours or more, 35 hours or more, 36 hours or more, 37 hours or more, 38 hours or more, 39 hours or more, 40 hours or more, 41 hours or more, 42 hours or more, 43 hours or more, 44 hours or more, 45 hours or more, 46 hours or more, 47 hours or more, or 48 hours It means to fast for more than.
  • the fasting period is determined by a doctor or the like in view of the subject's health condition, and is preferably set to a period equal to or longer than the time required for the subject to reach fasting blood sugar, for example.
  • the fasting period may be, for example, a period of time during which GLUT1 expression on the intravascular surface of cerebrovascular endothelial cells increases or reaches a plateau.
  • the fasting period can be, for example, the above period of 12 hours or longer, 24 hours or longer, or 36 hours or longer. Fasting may also be accompanied by other treatments that do not substantially affect blood glucose levels or GLUT1 expression on the intravascular surface.
  • blood glucose levels can be raised by various methods well known to those skilled in the art, for example, administration of substances that induce elevation of blood glucose levels, e.g., glucose, fructose (fructose), galactose, etc. It can be increased by administration of monosaccharides that induce blood sugar levels, administration of polysaccharides that induce blood sugar levels such as maltose, intake of carbohydrates that induce blood sugar levels such as starch, or meals.
  • blood sugar manipulation refers to controlling (or maintaining) a hypoglycemic state in a subject and then increasing the blood sugar level. After controlling the subject to a hypoglycemic state, the subject's blood glucose level can be maintained hypoglycemic.
  • the time to maintain the blood sugar level of the subject at hypoglycemia is, for example, 0 hours or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more , 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, 19 hours or more, 20 hours or more, 21 hours or more, 22 hours or more, 23 hours or more, 24 hours or more, 25 hours or more, 26 hours or more, 27 hours or more, 28 hours or more, 29 hours or more, 30 hours or more, 31 hours or more, 32 hours or more, 33 hours or more , 34 hours or more, 35 hours or more, 36 hours or more, 37 hours or more, 38 hours or more, 39 hours or more, 40 hours or more, 41 hours or more, 42 hours or more, 43 hours or more, 44 hours or more, 45 hours or more
  • Blood sugar levels can then be raised.
  • “maintaining blood sugar” means, for example, taking other drugs or drinking beverages such as water, as long as the goal of maintaining hypoglycemia in the subject is achieved. Inducing hypoglycemia may be accompanied by other treatments that do not substantially affect blood glucose. Hypoglycemia is preferably not pathologic hypoglycemia, but may mean having sufficient blood sugar to remain conscious, eg, fasting blood sugar levels. Hypoglycemia may be hypoglycemia at a level necessary to facilitate crossing of the BBB by a modified antibody displaying a GLUT1 ligand, which is subsequently manipulated to raise blood sugar.
  • a carrier whose outer surface is modified with a GLUT1 ligand shows accumulation in the brain even when administered to a subject (see WO2015/075942A).
  • dosing regimens according to the invention may not induce fasting or hypoglycemia and/or may not induce elevated blood glucose levels.
  • a carrier whose outer surface is modified with glucose such that glucose is exposed on the surface specifically a vesicle such as a micelle or a polyion complex polymersome (PICsome)
  • these carriers are remarkably enhanced. It is delivered into the brain (brain parenchyma) across the blood-brain barrier (see WO2015/075942A).
  • dosing regimens according to the present invention preferably comprise administering the composition to a subject who is fasted or hypoglycemic induced, but more preferably dosing regimens according to the present invention include fasting or hypoglycemia-induced subjects. or administering the composition to a subject that has induced hypoglycemia and inducing an increase in blood glucose levels in the subject.
  • the composition may be administered to the subject concurrently, sequentially or sequentially with the induction of elevated blood glucose levels in the subject.
  • a dosing regimen may or may not have an interval between administering the composition to the subject and inducing an increase in blood glucose levels in the subject.
  • the composition When the composition is administered at the same time as inducing an increase in blood glucose level in the subject, the composition may be administered to the subject in a form mixed with an agent that induces an increase in blood glucose level. , may be administered in a form separate from the agent that causes the induction of elevated blood glucose levels in the subject. Also, the composition induces an increase in blood glucose levels in the subject and, if administered to the subject sequentially or sequentially, the composition prior to inducing an increase in blood glucose levels in the subject. It may be administered to the subject on or after, but preferably the composition is administered to the subject prior to inducing an increase in blood glucose levels in the subject.
  • the composition is administered to the subject within, within 15 minutes, or within 10 minutes.
  • an increase in blood glucose level is induced in said subject within, within 1 hour, within 45 minutes, within 30 minutes, within 15 minutes or within 10 minutes. Two or more cycles of the above regimen may be performed.
  • the context of glucose administration and sample administration can be determined by the timing of crossing the blood-brain barrier.
  • Glucose administration in the present invention can be replaced with dietary intake. According to WO2015/075942A, it is also revealed that micelles pass through the BBB and accumulate in the brain without glycemic manipulation. In some aspects of the invention, the subject has not been induced to be hypoglycemic. In some aspects of the invention, the subject has been induced to be hypoglycemic, but not induced to have elevated blood glucose levels.
  • a modification may reduce or abolish (loss) the binding affinity of the antibody for antigen. By doing so, it can be expected that the function of the antibody in non-target tissues will be reduced, and the occurrence of side effects and adverse events will be reduced.
  • This reduction in binding affinity is preferably greater, e.g. or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.1% or less.
  • Binding affinities can be measured, for example, in serum or in saline.
  • the antibodies of the invention can be inactivated by modification. That is, the antibodies of the present invention can be inactivated antibodies.
  • the modification may be site-specific and environmentally responsive.
  • the modification may be a reducing environment responsive modification.
  • the antibody can be cleaved in the reducing environment of the brain parenchyma or tumor tissue, thereby partially or fully restoring (or reactivating) the antibody's binding affinity for the antigen. be able to.
  • Restoration or reactivation is preferably greater, e.g., the binding affinity of the restored or reactivated antibody is 50% or more, 55% or more, 60% or more, 65% of that of the unmodified antibody Greater than or equal to 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, or 95% or greater.
  • the antibodies of the present invention are characterized by modifications that are environmentally responsive, particularly reducing environmentally responsive, that are cleaved in an environmentally dependent manner to restore or reactivate the binding affinity of the antibody for its antigen.
  • the antibodies of the present invention can be environmentally responsive reactivatable antibodies.
  • the modification to the antibody may further comprise modification by a targeting molecule to a specific molecule, and present the targeting molecule to the specific molecule. This allows the antibody to be targeted to a specific molecule.
  • the specific molecule is immunologically distinguishably different from the antigen of the antibody. That is, the antibodies of the present invention can be targeting antibodies capable of targeting molecules that are immunologically distinguishable from antigens.
  • a modification to an antibody may further comprise a modification by a targeting molecule to a specific molecule, presenting the targeting molecule to the specific molecule, And the modification is one that reduces or abolishes the binding affinity of the antibody for antigen.
  • an antibody of the invention has been modified to reduce or eliminate its binding affinity for its antigen.
  • the modifications are additionally endowed with the ability to target specific antigens and deliver antibodies to the target antigen site.
  • the modification may be a site-specific environmentally responsive modification, in particular the modification may be a reducing environmentally responsive modification.
  • the antibody of the present invention is, for example, an antibody that has the ability to target a molecule that is immunologically distinguishable from an antigen, is an inactivated antibody, and is capable of environmental responsive reactivation. It can be an antibody. Reduction or loss of binding affinity of antibodies for antigens can be caused by steric hindrance by uncharged hydrophilic polymer blocks. Therefore, the binding affinity of the antibody for the antigen can be reduced or eliminated to a greater extent by increasing the bulk of the uncharged hydrophilic polymer block and/or by increasing the modification rate.
  • Inactivated antibodies can be achieved by modification of antibody amino groups with uncharged hydrophilic polymer blocks (eg, polyethylene glycol blocks and polyoxazoline blocks).
  • the non-charged hydrophilic polymer block can enhance the biocompatibility and blood retention of the antibody, and improve the ability of the antibody to migrate into the target tissue.
  • the uncharged hydrophilic polymer block has high water solubility and can be spread around the antibody, thereby inhibiting the antibody's access to its antigen.
  • modification with uncharged hydrophilic polymer blocks eg, polyethylene glycol
  • modification with uncharged hydrophilic polymer blocks that can suppress the binding affinity of antibodies to their original antigens can be preferably used.
  • the environmentally responsive reactivatable antibody may have an environmentally cleavable linker interposed between the antibody and the uncharged hydrophilic polymer block.
  • Environmentally responsive reactivating antibodies respond to the environment of a particular tissue by cleaving the linker, releasing the uncharged hydrophilic polymer block from the antibody, liberating (or releasing) the antibody, and binding the antibody to its antigen. Restores or reactivates affinity.
  • Environmentally cleavable linkers have a reducing environmentally cleavable bond (eg, a disulfide bond) within the linker.
  • a targeting antibody can display a targeting molecule at the end of an uncharged hydrophilic polymer block (eg, polyethylene glycol block and polyoxazoline block) that modifies the amino groups of the antibody.
  • a targeting antibody can have a targeting molecule that targets the inactivating antibody to a specific tissue.
  • the antibody is, for example, a targeting antibody that targets a special environment in which disulfide bonds are cleaved.
  • the environment in which disulfide bonds are cleaved can be, for example, a reducing environment (eg, brain parenchyma or tumor tissue), or low pH conditions (eg, physiologically possible low pH conditions).
  • the antibody of the present invention can be an inactivated antibody and an environmentally responsive reactivatable antibody, for example, except under special circumstances in which disulfide bonds are cleaved.
  • Such antibodies are referred to herein as environmental targeting reactivatable antibodies.
  • the pharmaceutical compositions of the invention include environmental targeting reactivatable antibodies.
  • the antibody can be a brain-targeted targeting antibody, an inactivating antibody, and a reactivating antibody responsive to a reducing environment.
  • Such antibodies are referred to herein as brain-targeted reactivatable antibodies.
  • the antibody can be a tumor-targeting antibody, an inactivating antibody, and a reactivating antibody responsive to a reducing environment.
  • Such antibodies are referred to herein as tumor-targeting reactivatable antibodies.
  • a polyethylene glycol block modified at its ends with a targeting molecule is attached to an amino acid of an antibody via an eco-responsive bond (e.g., a reducing eco-responsive bond, e.g., a disulfide bond).
  • an antibody consists of an uncharged hydrophilic polymer block (e.g., a polyethylene glycol block) modified at its ends with a targeting molecule to form an environmentally responsive bond (e.g., a reduced environmentally responsive bond, e.g., a disulfide bond).
  • an environmentally responsive bond e.g., a reduced environmentally responsive bond, e.g., a disulfide bond
  • the antibody can be an antibody that binds to a cancer antigen.
  • the antibody can be an antibody that binds to any of the immune checkpoint molecules.
  • the antibody is LL1 (anti-CD74 antibody), LL2 or RFB4 (anti-CD22 antibody), veltuzumab (hA20, anti-CD20 antibody), rituxumab (anti-CD20 antibody), obinutuzumab (GA101, anti-CD20 antibody) , lambrolizumab (anti-PD-1 receptor antibody), nivolumab (anti-PD-1 receptor antibody), ipilimumab (anti-CTLA-4 antibody), RS7 (anti-epithelial glycoprotein-1 (EGP-1, also known as TROP-2 ), PAM4 or KC4 (both anti-mucin), MN-14 (anti-carcinoembryonic antigen (also known as CEA, CD66e or CEACAM5), MN-15 or MN-3 (anti-
  • hPAM4 U.S. Pat. No. 7,282,567
  • hA20 U.S. Pat. No. 7,251,164
  • hA19 U.S. Pat. No. 7,109,304
  • hIMMU-31 US Pat. No. 7,300,655
  • hLL1 US Pat. No. 7,312,3108
  • hLL2 US Pat. No. 7,074,403
  • hMu-9 US Pat. No. 7 , 387,773
  • hL243 US Pat. No. 7,612,180
  • hMN-14 US Pat. No. 6,676,924
  • hMN-15 US Pat. No.
  • target molecules include, for example, carbonic anhydrase IX, B7, CCCL19, CCCL21, CSAp, HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5 , CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L , CD44, CD45, CD46, CD47, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM6, CTLA-4 , ⁇ -fetoprotein (AFP), VEGF, fibronectin splic
  • the antibody can specifically bind to an antigen.
  • the antibody is 10 ⁇ 15 M or greater, 10 ⁇ 14 M or greater, 10 ⁇ 13 M or greater, 10 ⁇ 12 M or greater, 10 ⁇ 11 M or greater, or 10 ⁇ 10 M or greater to the antigen. or higher binding affinity.
  • the antibody is 10 ⁇ 8 M to 10 ⁇ 15 M, 10 ⁇ 9 M to 10 ⁇ 14 M, 10 ⁇ 10 M to 10 ⁇ 13 M, or 10 ⁇ 11 M to 10 ⁇ 1 M to the antigen. It may have a binding affinity of 12 M, or between any of the above upper and lower limits.
  • the modified antibody has a binding affinity for the antigen of 10 ⁇ 6 M or greater, 10 ⁇ 5 M or greater, 10 ⁇ 4 M or greater, 10 ⁇ 3 M or greater, 10 ⁇ 2 M or greater, or an undetectable binding affinity. can have gender.
  • the modified antibody has a concentration of 10 ⁇ 6 M to 10 ⁇ 1 M, 10 ⁇ 4 M to 10 ⁇ 1 M, 10 ⁇ 3 M to 10 ⁇ 1 M, or 10 ⁇ 2 M to 10 ⁇ 1 M to the antigen. It may have a binding affinity between -1 M or below the limit of detection. In certain aspects, the antibody has the binding affinity for the antigen of the antibody described above, and the modified antibody can have the binding affinity for the antigen of the modified antibody described above.
  • the targeting molecule can be a GLUT1 ligand. That is, the present invention provides an antibody modified with a GLUT1 ligand, wherein the GLUT1 ligand, an uncharged hydrophilic polymer block, an environment-responsive bond, and an antibody are linked in that order.
  • Environmentally responsive bonds are bonds that cleave under the reducing environment of brain parenchyma or tumor tissue. Environmentally responsive binding can be stable in blood.
  • the antibody of the present invention is a GLUT1-modified antibody, even when administered into the blood, wherein the GLUT1 ligand, the uncharged hydrophilic polymer block, the environment-responsive bond, and the antibody are This order maintains the form of existence of the ligated antibody. Then, upon internalization into the brain parenchyma or tumor tissue, the environmentally responsive bonds are cleaved to separate the uncharged hydrophilic polymer block from the antibody.
  • modifications may be made to the amino groups of the antibody.
  • an antibody in the form of a conjugate in which a GLUT1 ligand, an uncharged hydrophilic polymer block, an environment-responsive bond, and an antibody are linked in that order.
  • a modified antibody having a structure of uncharged hydrophilic polymer block-environmentally responsive bond-antibody is provided.
  • the symbol "-" represents a bond or spacer, and means that the element described before the symbol and the element described after the symbol are linked via a bond or spacer. Spacers have chemical properties that are stable in vivo.
  • the eco-responsive bond is a reduced eco-responsive bond, eg, a disulfide bond.
  • the GLUT1 ligand can be glucose. Glucose is linked to the uncharged hydrophilic polymer block so that it can bind to GLUT1.
  • Antibodies having amino groups modified with -C(O)-OL 1 -SSL 2 - (uncharged hydrophilic polymer blocks).
  • L 1 is optionally substituted lower alkylene (that is, substituted or unsubstituted lower alkylene), preferably ethylene.
  • L2 is a bond ( single bond) or a non-cleavable spacer (eg, a stable spacer).
  • the uncharged hydrophilic polymer block may or may not be further modified with a targeting molecule.
  • L 2 can be L 3 —O—C(O)—NH—L 4 .
  • Such antibodies can be cleaved at the disulfide bonds in a reducing environment to release the unmodified form of the antibody. According to the invention, the released unmodified antibody can regain its original binding affinity for the antigen.
  • L 1 , L 3 and L 4 are each independently a bond or optionally substituted lower alkylene ⁇ .
  • the antibody can be a reducing environment-responsive reactivatable antibody (eg, a brain-targeted reactivatable antibody).
  • the uncharged hydrophilic polymer block may or may not be further modified with a targeting molecule.
  • the targeting molecule can be a GLUT1 ligand.
  • the GLUT1 ligand can be glucose.
  • Glucose is linked to the uncharged hydrophilic polymer block so that it can bind to GLUT1.
  • Such antibodies can be cleaved at the disulfide bonds in a reducing environment to release the unmodified form of the antibody.
  • the released unmodified antibody can regain its original binding affinity for the antigen.
  • the antibodies are environmentally responsive, e.g., disulfide bonds (-S -S-). Cleavage of the disulfide bond releases the uncharged hydrophilic polymer block from the antibody, allowing the antibody to regain binding affinity for the antigen.
  • disulfide bonds e.g., disulfide bonds (-S -S-). Cleavage of the disulfide bond releases the uncharged hydrophilic polymer block from the antibody, allowing the antibody to regain binding affinity for the antigen.
  • the antibody can release an intact antibody (or unmodified or native antibody) by chain reaction after disulfide bond cleavage.
  • An intact antibody means that the modified amino groups have reverted to --NH 2 groups.
  • the released intact antibody can restore the original antibody's binding affinity for the antigen.
  • the antibody has reduced or lost binding affinity for its antigen by modification with an uncharged hydrophilic polymer block. In some embodiments, the antibody has a binding affinity for its antigen that is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 4% or less by modification with an uncharged hydrophilic polymer block. Below, it is reduced to 3% or less, 2% or less, 1% or less, 0.1% or less, or 0.01% or less. Antibodies that have reduced or lost binding affinity by modification with uncharged hydrophilic polymer blocks are inactive until the environmentally responsive bond is cleaved, thus functioning the antibody outside of the targeted site. can be prevented. In this way, the function of the antibody can be restored specifically to the target site.
  • Antibodies bind antigens in an environment where disulfide bonds are cleaved, e.g., in a reducing environment (e.g., the reducing environment of brain parenchyma or tumor tissue), or in low pH conditions (e.g., physiologically possible low pH conditions). sell.
  • a reducing environment e.g., the reducing environment of brain parenchyma or tumor tissue
  • low pH conditions e.g., physiologically possible low pH conditions.
  • the binding affinity (KD) for antigen is, for example, 10 ⁇ 8 M or less, 10 ⁇ 9 M or less, 10 ⁇ 10 M or less, 10 ⁇ 11 M or less, or 10 ⁇ 12 M or less. could be.
  • Antigens present in environments where disulfide bonds are cleaved are associated with cancers, e.g. It can be an antigen, or an immune checkpoint molecule.
  • An immune checkpoint molecule means a molecule involved in an immune checkpoint. Immune checkpoint molecules bind to their counterparts (ie, binding partners immune checkpoint molecules) to activate immune checkpoints and suppress immune action.
  • the immune checkpoint molecule can be, for example, a molecule involved in the PD-1 system immune checkpoint.
  • Molecules involved in the PD-1 immune checkpoint include programmed cell death-1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T Cellular immunoglobulin domain and mucin domain-3 (T-cell immunoglobulin domain and mucin domain-3, or TIM-3), lymphocyte activation gene 3 (LAG-3), and type V immunoglobulin domain-containing suppressor of T-cell activation (V-type immunoglobulin domain-containing suppressor of T-cell activation, or VISTA).
  • the immune checkpoints responsible for each are called PD-1 immune checkpoint, CTLA-4 immune checkpoint, TIM-3 immune checkpoint, LAG-3 immune checkpoint, and VISTA immune checkpoint.
  • An immune checkpoint inhibitor can inhibit the function of an immune checkpoint, eg, by binding to an immune checkpoint molecule or its counterpart. For example, by inhibiting the binding of PD-1 to PD-L1 or PD-L2, the PD-1 system immune checkpoint can be inhibited. Also, by inhibiting the binding of CTLA-4 to CD80 or CD86, the CTLA-4 system immune checkpoint can be inhibited. In addition, by inhibiting the binding of TIM-3 to galectin-9, the TIM-3 system immune checkpoint can be inhibited. In addition, by inhibiting the binding of LAG-3 to MHC class II molecules, the LAG-3 system immune checkpoint can be inhibited.
  • the VISTA system immune checkpoint can be inhibited.
  • one or more selected from the group consisting of PD-1-based immune checkpoint, CTLA-4-based immune checkpoint, TIM-3-based immune checkpoint, Lag3-based immune checkpoint, and VISTA-based immune checkpoint can inhibit the immune checkpoint of Antibodies that inhibit the binding of two proteins can bind to immune checkpoint molecules or their counterparts.
  • antibodies that inhibit the PD-1 system immune checkpoint are antibodies selected from the group consisting of anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies (e.g., nivolumab, pemprolizumab, avelumab, atezolizumab, and durvalumab).
  • Antibodies that inhibit CTLA-4-based immune checkpoints can also be antibodies selected from the group consisting of anti-CDLA-4 antibodies, anti-CD80 antibodies, and anti-CD86 antibodies (eg, ipilimumab and tremelimumab).
  • the antibody that inhibits the TIM-3-based immune checkpoint can be an antibody (eg, MGB453) selected from the group consisting of anti-TIM-3 antibodies and anti-galectin-9 antibodies.
  • the antibody that inhibits the VISTA-based immune checkpoint can be an antibody (eg, JNJ-61610588) selected from the group consisting of anti-VISTA antibody and anti-VSIG-3/IGSF11 antibody.
  • the antibodies used in the present invention bind to immune checkpoint molecules (eg, PD-1, CTLA4, LAG-3, TIGIT, VISTA and TIM-3) expressed on immune cells.
  • the antibodies used in the present invention are immune checkpoint molecule counterparts expressed on immune cells (e.g., PD-L1, PD-L2, CD80, CD86, galectin-9, MHC class II molecule, VSIG-3/IGSF11).
  • the PD-1 based immune checkpoint can be inhibited by blocking the binding of PD-1 and PD-L1.
  • the PD-1 system immune checkpoint can be inhibited by blocking the PD-1 signal.
  • the antibodies of the invention can be immune checkpoint inhibitors.
  • Antibodies of the invention can bind to any of the immune checkpoint molecules and block binding to their counterpart immune checkpoint molecules.
  • Antibodies of the invention can be PD-1 system immune checkpoint inhibitors.
  • An antibody of the invention can be, for example, an antibody capable of blocking the binding of PD-1 and PD-L1.
  • An antibody of the invention can be, for example, an antibody that binds to PD-1 (or an anti-PD-1 antibody).
  • An antibody of the invention can be, for example, an antibody that binds to PD-L1 (or an anti-PD-L1 antibody).
  • Antibodies of the invention can be anti-PD-L1 antibodies capable of blocking the binding of PD-1 and PD-L1.
  • Antibodies of the invention can be, for example, anti-PD-1 antibodies capable of blocking the binding of PD-1 and PD-L1.
  • Blocking can mean inhibiting binding by 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more.
  • a bloodstream-stable spacer means a spacer that is stable to the extent that it can be stably present in the blood over the time required for its transition from administration to target tissue (for example, brain parenchyma or tumor tissue) in the bloodstream.
  • a bond selected from the group consisting of a carbon-carbon bond, an amide bond, a phosphodiester bond, an ester bond, an ether bond, an alkylene, a carbamate bond, a thiocarbamate bond, a thioester bond, a thioether bond, a disulfide bond, and combinations thereof It can be a flow-stable spacer.
  • a bloodstream stable spacer is pharmaceutically acceptable.
  • the uncharged hydrophilic polymer segment and the antibody can be linked by these spacers.
  • spacers that are stable in the bloodstream include, but are not limited to, substituted or unsubstituted alkylene or substituted or unsubstituted heteroalkylene. Whether a spacer is stable in the blood stream can be determined by assessing the stability of the spacer in, for example, isolated blood or serum-containing saline. A person skilled in the art can appropriately determine the time required from administration to target tissue (eg, brain parenchyma or tumor tissue).
  • target tissue eg, brain parenchyma or tumor tissue.
  • the time required for the transition from administration to the target tissue is, for example, 1 hour or longer, 2 hours or longer, 3 hours or longer, 4 hours or longer, 5 hours or longer, 6 hours or longer, and 7 hours. 8 hours or more, 9 hours or more, 10 hours or more, 12 hours or more, 15 hours or more, 18 hours or more, 21 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, It can be 6 days or more, or 7 days or more.
  • the time required to transition from administration to the target tissue is, for example, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less. can be days or less.
  • the time required for transit from administration to target tissue can be, for example, from 1 hour or more to 1 day or less.
  • an antibody modified with uncharged hydrophilic polymer segments the antibody being able to bind to an antigen, in blood in the form of an antibody modified with uncharged hydrophilic polymer segments
  • the antibody is provided in a form dissociated from the uncharged hydrophilic polymer segment after being transferred from the blood vessel to a special environment where disulfide bonds are cleaved (e.g., brain parenchyma or tumor tissue).
  • the antibody dissociates from the uncharged hydrophilic polymer segments upon passage through vascular endothelial cells into a special environment where disulfide bonds are cleaved.
  • the antibody dissociates with uncharged hydrophilic polymer segments upon entering tumor tissue, within tumor tissue, or upon entering the brain parenchyma, during transcytosis of the BBB, or in the brain parenchyma.
  • the antibody is linked to an uncharged hydrophilic polymer segment with a reducing environment-responsive linker, which bond is cleaved under a reducing environment within the target tissue (e.g., brain parenchyma or tumor tissue).
  • the reducing environment-responsive bond can be a disulfide bond.
  • antibodies dissociated with uncharged hydrophilic polymer segments have a stronger binding affinity for antigen than antibodies modified with uncharged hydrophilic polymer segments.
  • the antibody modified with uncharged hydrophilic polymer segments has a weaker binding affinity to antigen than the antibody before modification (unmodified antibody).
  • an antibody modified with a non-charged hydrophilic polymer segment has a weaker binding affinity to an antigen than an antibody before modification (unmodified antibody), and the non-charged hydrophilic polymer
  • Antibodies with dissociated segments have a stronger binding affinity for antigen than antibodies modified with uncharged hydrophilic polymer segments.
  • antibodies modified with uncharged hydrophilic polymer segments are cleaved with said segments in special environments where disulfide bonds are cleaved, e.g. within brain parenchyma or tumor tissue, and the modification reduces It can restore the binding properties of the antibody that had been lost.
  • Such antibodies can be used without special manipulations (e.g., glycemic manipulation) in patients with diseases in which vascular endothelial cells are weakened or the blood-brain barrier is weakened. It can be useful because it can reach the environment (eg, brain parenchyma or tumor tissue) and restore its binding properties within the environment (brain parenchyma or tumor tissue) without modification by .
  • said uncharged hydrophilic polymer segment may be modified with a GLUT1 ligand.
  • Modification with a GLUT1 ligand can target the antibody within endosomes of vascular endothelial cells (e.g., brain vascular endothelial cells) or in target tissues (e.g., brain parenchyma or It is useful from the viewpoint of sending it into the tumor tissue).
  • vascular endothelial cells e.g., brain vascular endothelial cells
  • target tissues e.g., brain parenchyma or It is useful from the viewpoint of sending it into the tumor tissue.
  • an antibody modified with an uncharged hydrophilic polymer via a linker wherein the uncharged hydrophilic polymer is modified with a GLUT1 ligand.
  • the antibody can pass through the blood vessels of tumor tissue and enter into tumor tissue due to the EPR effect or the like.
  • the antibody can bind to GLUT1 expressed on the luminal surface of vascular endothelial cells (eg, cerebral vascular endothelial cells) via GLUT1 ligands.
  • vascular endothelial cells e.g., cerebral vascular endothelial cells
  • endocytosis When glucose is administered (or GLUT1 ligand is administered) to a hypoglycemic subject, substances bound to the luminal surface of vascular endothelial cells (e.g., cerebral vascular endothelial cells) of the subject undergo endocytosis. It is taken up into vascular endothelial cells and at least part of it is delivered by transcytosis into a special environment (eg brain parenchyma) where disulfide bonds are cleaved.
  • vascular endothelial cells e.g., cerebral vascular endothelial cells
  • GLUT1 is expressed on the luminal surface of vascular endothelial cells (e.g., cerebral vascular endothelial cells) of a subject whose blood sugar is lowered, and the antibody of the present invention binds to GLUT1 via a GLUT1 ligand, (or a GLUT1 ligand) is taken up by endocytosis into vascular endothelial cells, at least a portion of which is delivered by transcytosis into the environment (eg, brain parenchyma).
  • the binding of GLUT1 to the antibody can be confirmed by an assay that evaluates the binding of isolated GLUT1 to the antibody in an in vitro experiment.
  • Uncharged hydrophilic polymers include, for example, polyethylene glycol (PEG) and uncharged hydrophilic polymers such as polyoxazolines.
  • uncharged means that the charge is neutralized throughout the polymer segment.
  • Uncharged hydrophilic polymers are biocompatible.
  • the antibody of the invention is an uncharged hydrophilic polymer segment modified with a ligand (second ligand) for a receptor in a target tissue (e.g., brain parenchyma or tumor tissue) other than the GLUT1 ligand or not.
  • the linkers in the above antibodies of the present invention can be linked to side chain amino groups of lysine residues of the antibody.
  • the linkage may preferably be a covalent bond.
  • the lysine residues that join the linkers can be present in the heavy and/or light chain variable regions of the antibody.
  • the lysine residues that join the linkers can be present within the CDR regions of the heavy and/or light chain variable regions of the antibody.
  • Modification with a targeting molecule e.g. GLUT1 ligand
  • a targeting molecule e.g. GLUT1 ligand
  • a special environment e.g. brain parenchyma or tumor tissue
  • the antibody is transferred to a special environment where the disulfide bond is cleaved (e.g., brain parenchyma or tumor tissue).
  • the PEG modified by the antibody-modifying targeting molecule e.g., GLUT1 ligand
  • modification of the side chain amino groups of lysine residues of antibodies can reduce the binding properties of antibodies depending on the strength of the modification.
  • the PEG modified by the antibody-modifying targeting molecule e.g., GLUT1 ligand
  • the antibody-modifying targeting molecule may be A targeting molecule (eg, a GLUT1 ligand) may modify PEG.
  • a targeting molecule eg, a GLUT1 ligand
  • a particular environment in which disulfide bonds are cleaved can be, for example, brain parenchyma or tumor tissue. For example, brain parenchyma or tumor tissue has a reducing environment.
  • the reducing environment of brain parenchyma or tumor tissue is a reducing environment of strength equivalent to 2 mM glutathione (GSH) aqueous solution.
  • the linker can be configured such that the linker is cleaved after the antibody is delivered to the brain parenchyma or tumor tissue by including a cleavage site in the linker that can be cleaved under a reducing environment.
  • Such linkers are referred to as cleavable linkers in a reducing environment.
  • the present invention provides antibodies modified by an uncharged hydrophilic polymer (eg, PEG) modified with a targeting molecule (eg, a GLUT1 ligand) via a linker that is cleavable in a reducing environment.
  • Antibodies of the invention are preferably capable of cleaving the linker in brain parenchyma or tumor tissue that provides a reducing environment.
  • a linker that is cleavable under a reducing environment can, for example, have a disulfide bond as the cleavage site.
  • a targeting molecule e.g., GLUT1 ligand
  • the linker is cleaved after the antibody has been delivered to the brain parenchyma or tumor tissue. and can release antibodies within the brain parenchyma or tumor tissue.
  • a linker that is cleavable in a reducing environment can be configured such that cleavage results in side chain amino groups of lysine residues of the antibody to unsubstituted amino groups.
  • a linker (antibody-NH)-CO-O-C 2 H 4 -SSL 2 -(uncharged hydrophilic polymer) ⁇ wherein L 2 is a binding or blood-stable is a linker ⁇
  • L 2 is a binding or blood-stable is a linker
  • antibodies of the invention in which the GLUT1 ligand is glucose (Gluc), the uncharged hydrophilic polymer is polyethylene glycol (PEG), and L 2 is —C 2 H 4 —O—CO— are tested in a reducing environment. shows the mechanism of dissociating from the linker and returning to the original antibody. This mechanism is similar when the GLUT1 ligand is a GLUT1 ligand other than glucose and the uncharged hydrophilic polymer is an uncharged hydrophilic polymer other than PEG.
  • a linker cleavable in a reducing environment is configured such that cleavage dissociates the linker moiety from the side chain amino group of the lysine residue of the antibody and the amino group reverts to an unsubstituted amino group, thereby releasing the target tissue. (eg, in brain parenchyma or tumor tissue), the antibody can be returned to its undisplaced state. It has been pointed out that the binding properties of antibodies can be reduced by modifying the side chain amino groups of lysine residues, and modification of the side chain amino groups of lysine residues reduces the binding properties of antibodies.
  • the side chain amino group of the lysine residue of the antibody is unmodified and unsubstituted. to allow the antibody to regain its binding properties.
  • the absolute zeta potential of the modified antibody is 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the absolute zeta potential of the unmodified antibody. obtain. In some embodiments, the absolute zeta potential of the modified antibody can be on the order of 3% to 1% of the absolute zeta potential of the unmodified antibody. A modified antibody can have a reduced absolute zeta potential because it is modified with an uncharged hydrophilic polymer block.
  • the modified antibody is greater in mean hydrodynamic diameter (volume average) than the unmodified antibody, e.g. or greater than 70%. In some embodiments, the modified antibody has a mean hydrodynamic diameter (volume average) that is 40% to 80%, or 50% to 70% larger than the unmodified antibody.
  • the binding of the modified antibody to the antigen is 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6 % or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.
  • the GLUT1 ligand can be glucose.
  • glucose can be linked to PEG via, for example, its 2-, 3- or 6-position carbon atoms and can favorably interact with GLUT1.
  • PEG can have a Mw (weight average molecular weight) of 2,000 to 12,000, for example, 5,000.
  • 10-90%, 20-80%, 30-70%, or 40-60% of the side chain amino groups (primary amines) of lysine residues are polyethylene glycol (PEG) can be modified through a linker by
  • PEG polyethylene glycol
  • the proportion of PEG-modified amino groups among the side chain amino groups of lysine residues may be indicated by adding the proportion (%) after "PEG".
  • the antibody of the present invention may be modified with both GLUT1 ligand-modified PEG and non-GLUT1 ligand-modified PEG.
  • the PEGs that modify the antibodies for example, 10-100%, 30-80%, or 40-60% of the PEGs can be modified with GLUT1.
  • the proportion of PEG modified with a GLUT1 ligand may be indicated by adding the proportion (%) after "G” or "Gluc".
  • the antigen of the antibody When the antigen of the antibody is a surface antigen of a tumor within the target tissue (e.g., brain parenchyma) or a surface antigen of a cell within the tumor tissue, the antibody exhibits antibody-dependent cellular cytotoxicity (ADCC activity) and/or complement Antibodies with dependent cytotoxic activity (CDC activity) can be used. ADCC activity can be enhanced, for example, by subclassing the antibody to IgG1.
  • the antigen of the antibody is a surface antigen of a tumor within the target tissue (e.g., brain parenchyma) or a surface antigen of a cell within the tumor tissue
  • the antibody When the antigen of the antibody is a surface antigen of a tumor within the target tissue (e.g., brain parenchyma) or a surface antigen of a cell within the tumor tissue, the antibody is in the form of an antibody-drug conjugate (ADC) with a cytotoxic agent.
  • ADC antibody-drug conjugate
  • An antibody in certain aspects, can be in the form of an antibody-drug conjugate (ADC).
  • the linker modifies the antibody portion.
  • the modification is of sufficient length or size to envelop the linker portion of the ADC and may stabilize the linker from degradation or inhibit degradation of the linker by components in vivo.
  • the modification may also be of sufficient length or size to envelop the linker portion and drug portion of the ADC, exposing the drug portion of the ADC and reducing side effects from interacting with other components.
  • ADCs containing modified antibody moieties have linker moieties and drug moieties per 1 ADC, but are not particularly limited, for example, about 1 to 10, 2 to 8, 2 to 5, 2 to 4, or 3 to 4 can contain.
  • ADCs comprising modified antibody moieties are 12 nm or greater, 13 nm or greater, 14 nm or greater, 15 nm or greater, 16 nm or greater, 17 nm or greater, about 18 nm or less, and/or 30 nm or less, 29 nm or less, 28 nm or less, 27 nm or less, 26 nm or less, or 25 nm. Below, for example, it may have an average hydrodynamic radius (volume average) of 12-30 nm, or 15-25 nm.
  • ADCs include, but are not limited to, gemtuzumab ozogamicin (Milotarg), ibritumomab tiuxetan (Zevalin), brentuximab vedotin (ADCETRIS), trastuzumab emtansine (Kadcyra), inotuzumab ozogamicin (Vesponsa), moxetumomab pasudotox-tdfk (LUMOXITI), polatuzumab It may contain one or more selected from the group consisting of vedotin-Piiq (Polivy), trastuzumab deruxtecan (Enherts), and enfortumab vedotin (PADCEV).
  • gemtuzumab ozogamicin Milotarg
  • ibritumomab tiuxetan Zevalin
  • the antibody of the present invention may be linked to an imaging agent.
  • Imaging agents include, for example, biocompatible fluorescent dyes (e.g., fluorescent dyes that emit fluorescence in the visible light region or near-infrared region) and luminescent dyes (e.g., luciferase), and radioisotopes, ultra Imaging agents such as acoustic probes, MRI contrast agents and CT contrast agents are included.
  • a conjugate of an imaging agent and an antibody can be appropriately prepared by those skilled in the art.
  • the antibodies of the present invention may be linked to physiologically active substances (eg, enzymes and nucleic acids). This can deliver the bioactive agent to the target tissue (eg, brain parenchyma or tumor tissue).
  • physiologically active substances eg, enzymes and nucleic acids
  • a conjugate of a physiologically active substance and an antibody can be appropriately prepared by those skilled in the art.
  • Brain diseases include, for example, brain tumors.
  • Brain tumors include, for example, brain tumors selected from the group consisting of glioma, glioblastoma, central nervous system primary malignant lymphoma, meningioma, pituitary adenoma, schwannoma, and craniopharyngioma.
  • Antibodies also include antibodies that bind to PD-L1 expressed on these tumors and neutralize its activity.
  • Antibodies include those that bind to PD-1 expressed on lymphocytes of these tumors and neutralize its activity.
  • Antibodies include those that bind to CTLA4 expressed on lymphocytes of these tumors and neutralize its activity.
  • treatment of disease means prevention of disease and cure of disease. Disease prevention is used in the sense of preventing the onset of disease, delaying onset, and reducing the onset rate. Treatment of a disease is meant to include slowing the rate of exacerbation of the disease, delaying exacerbation, preventing exacerbation, alleviating the symptoms of the disease, curing the disease, and remission of the disease.
  • the present invention also provides pharmaceutical compositions for treating or preventing disease (eg, cancer) in target tissues, comprising the antibodies of the present invention. Cancers include lung cancer (e.g.
  • lung squamous cell carcinoma lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer), head and neck cancer, hepatocellular carcinoma, renal cell carcinoma, colon cancer, melanoma , bladder cancer, ovarian cancer, gastric cancer, Merkel cell carcinoma, breast cancer (eg, triple-negative breast cancer), and Merkel cell carcinoma.
  • Tumors also include Hodgkin's lymphoma.
  • Antibodies or pharmaceutical compositions of the invention may be administered according to a dosing regimen.
  • the dosing regimen is lowering blood sugar in a subject, and thereafter, Inducing an increase in blood glucose levels in the subject such that more antibodies are translocated to target tissues (e.g., brain parenchyma) compared to not inducing an increase in blood glucose levels, and the present invention administering an antibody or pharmaceutical composition of
  • the subject has a blood-brain barrier or has a dysfunctional blood-brain barrier.
  • a dysfunctional blood-brain barrier allows macromolecules, including antibodies, to drop into the brain parenchyma.
  • subjects with a compromised blood-brain barrier may be administered modified antibodies of the invention without this dosing regimen.
  • the composition may be administered to the subject simultaneously, sequentially, or sequentially with the induction of elevated blood glucose levels in the subject.
  • a dosing regimen may or may not have an interval between administering the composition to the subject and inducing an increase in blood glucose levels in the subject.
  • the composition may be administered to the subject in a form mixed with an agent that induces an increase in blood glucose level.
  • the composition may be administered in a form separate from the agent that causes the induction of elevated blood glucose levels in the subject.
  • the composition induces an increase in blood glucose levels in the subject and, if administered to the subject sequentially or sequentially, the composition prior to inducing an increase in blood glucose levels in the subject. It may be administered to the subject on or after, but preferably the composition is administered to the subject prior to inducing an increase in blood glucose levels in the subject. within 1 hour, within 45 minutes, or 30 minutes after inducing an increase in blood glucose level in the subject, when an increase in blood glucose level is induced in the subject prior to administration of the composition to the subject Preferably, the composition is administered to the subject within, within 15 minutes, or within 10 minutes.
  • an increase in blood glucose level is induced in the subject after administration of the composition to the subject, within 6 hours, within 4 hours, or 2 hours after administration of the composition to the subject
  • an increase in blood glucose level is induced in said subject within, within 1 hour, within 45 minutes, within 30 minutes, within 15 minutes or within 10 minutes. Two or more cycles of the above regimen may be performed.
  • the context of glucose administration and sample administration can be determined by the timing of crossing the blood-brain barrier.
  • the GLUT1 ligand can be replaced with a molecule that binds to the target antigen.
  • a target antigen can be, for example, a membrane protein expressed on the cell surface. By doing so, the antibody can be concentrated on the surface of specific cells.
  • the antigen in one example, can be an antigen expressed on cells of the target tissue (eg, cells of the brain).
  • Molecules that bind to the target antigen include, for example, proteins that bind to the target antigen (e.g., antibodies or cyclic proteins), aptamers that bind to the target antigen, and lectins that bind to the target antigen (when the target antigen has sugar chains). Those skilled in the art can appropriately select or create and use them as molecules that bind to the target antigen of the present invention.
  • compositions of the present invention may further contain pharmaceutically acceptable excipients in addition to the antibody of the present invention.
  • Pharmaceutical compositions of the present invention can be in various forms, such as liquids (eg, injections), dispersions, suspensions, tablets, pills, powders, suppositories, and the like.
  • the pharmaceutical composition of the present invention is an injection and can be administered parenterally (eg, intravenously, transdermally, and intraperitoneally).
  • a method comprising administering the antibody of the present invention to a subject.
  • a method of delivering an antibody to a target tissue e.g., brain parenchyma or tumor tissue
  • administering to said subject an antibody of the invention or a pharmaceutical composition comprising said antibody.
  • a method is provided.
  • Administration can be intravenous.
  • antibodies of the invention can be administered according to dosing regimens according to the invention.
  • methods for modifying antibodies and methods for obtaining modified antibodies of the invention are provided, respectively.
  • luciferase-tagged mouse glioblastoma cell line-GL261 and luciferase-tagged CT2A were obtained from the JCRB cell bank (Osaka, Japan).
  • GL261 and CT2A cells were maintained in RPMI 1640 medium (Gibco; Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 U ml ⁇ 1 penicillin (Invitrogen) and 100 U ml ⁇ 1 streptomycin (Invitrogen).
  • Cells were cultured in a 37° C. incubator (Thermo Fisher Scientific) in an atmosphere of 5% CO 2 and 90% relative humidity.
  • Avelumab (trade name BAVENCIO), a clinically approved human anti-PD-L1 antibody (aPD-L1), is available from Merck & Co. was commercially obtained from Antibodies used for immunostaining are CD3 (BioLegend; catalog number 100205, clone: 17A2), CD4 (BioLegend; catalog number 100434, clone: GK1.5), CD45 (BD Biosciences; catalog number 553080, clone: 30-F11 ), CD8a (BD Biosciences; Catalog No. 553035, Clone: 53-6.7), FOXP3 (BD Biosciences; Catalog No. 560408, Clone: MF23), CD44 (BD Biosciences; Catalog No.
  • MeO-PEG-NH 2 (1.0 g, 0.167 mmol) or DIG-PEG-NH 2 (1.0 g, 0.167 mmol) dissolved in dichloromethane (DCM) was added to NPC- (CH 2 ) 2 --S—S—(CH 2 ) 2 --NPC (324 mg, 0.667 mmol) was added under an ice bath for 10 minutes. Pyridine (54 ⁇ L, 0.667 mmol) was added dropwise to the cold mixture with vigorous stirring. The reaction mixture was then stirred overnight at room temperature and subsequently dialyzed against DMSO using a MWCO 3.5 kDa membrane.
  • DCM dichloromethane
  • MeO-PEG-NH-C(O)-O-(CH 2 ) 2 -SS-(CH 2 ) 2 -NPC and DIG-PEG-NH-C(O)-O-(CH 2 ) 2 -SS-(CH 2 ) 2 -NPC was lyophilized for further use.
  • the resulting solution was then dialyzed sequentially against water, 10 mM HCl, and distilled water. Finally, the purified solution was lyophilized and the 1 H-NMR of the resulting polymer was measured in D 2 O at 25°C. The resulting polymer was Gluc-PEG-NH-C(O)-O-(CH 2 ) 2 -SS-(CH 2 ) 2 -NPC with DIG deprotected to glucose.
  • NPC-CH 2 ) 2 -CH 2 -CH 2 -CH 2 -(CH 2 ) 2 -NPC which forms non-cleavable crosslinks
  • MeO-PEG- NH-C(O)-O-( CH2 ) 2 - CH2 - CH2 -NPC and DIG-PEG-NH-C(O)-O-( CH2 ) 2 - CH2 - CH2 - CH2 —(CH 2 ) 2 —NPC was prepared.
  • Gluc-S-aPD-L1 with various glucose densities 100%:0%, 50%:50%, 25%:75%, 0%:100% in phosphate buffer (pH 8.4, 20 mM)
  • Gluc-PEG-(CH 2 ) 2 -SS-(CH 2 ) 2 -NPC (10.0 mg mL ⁇ 1 )
  • MeO-PEG-(CH 2 ) 2 -S dissolved at various feed ratios containing
  • a 1.0 mL solution containing -S-(CH 2 ) 2 -NPC (10.0 mg mL -1 ) was mixed with aPD-L1 (1.0 mg) and covalently coupled overnight at room temperature. rice field.
  • PEG-S-NPC Modification of an antibody with PEG-NH-C(O)-O-(CH 2 ) 2 -S-S-(CH 2 ) 2 -NPC (hereinafter sometimes referred to as PEG-S-NPC) is can be performed at an intensity that significantly reduces or loses its binding affinity.
  • Gluc-S-aPD-L1 Size distribution and zeta potential of aPD-L1 and Gluc-S-aPD-L1 were determined at pH 7.4 using a Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, UK). was evaluated by DLS measurements at 25 °C in 10 mM phosphate buffer. To determine the percent PEG modification, fluorescamine (30.0 ⁇ L, 3.0 mg mL ⁇ 1 ) dissolved in acetone was added to Gluc-S-aPD-L1 (90 ⁇ L, 1.0-10.0 ⁇ g mL ⁇ 1 ).
  • Circular dichroism spectra of aPD-L1 and Gluc-S-aPD-L1 incubated in the presence or absence of GSH (1.0 mM) were analyzed using a CD spectrometer (JASCO J-815). Measured at room temperature.
  • aPD-L1 and Gluc-S-aPD-L1 were preincubated in the presence or absence of GSH (1.0 mM) for various times to cleave disulfide bonds prior to reaction with PD-L1 protein.
  • the rate of recovery of activity was calculated using the above system.
  • aPD-L1 and Gluc-S-aPD-L1 were stained with CellMask TM Green Plasma Membrane Stain, aPD-L1 and Gluc-S-aPD-L1 treated with or without GSH (1.0 mM) were incubated with GL261 cells for an additional hour and finally analyzed by confocal laser scanning microscopy (LSM 880, Zeiss ) was used to assess the binding affinity of the antibodies.
  • GL261 tumor cells were treated with or without GSH (1.0 mM). aPD-L1 and Gluc-S-aPD-L1 for 1 hour and washed 3 times. APC-labeled anti-mouse PD-L1 antibody was then further incubated with GL261 cells for 1 hour, followed by analysis using the FlowJo software package to measure residual PD-L1 protein.
  • aPD-L1 and Gluc-S-aPD-L1 with different glucose densities were administered to healthy BALB/c mice via the tail vein at a dose of 1.0 mg kg ⁇ 1 .
  • Blood samples were taken at 1 hour, 4 hours, 12 hours and 24 hours after dosing. Blood samples were extracted and treated with or without GSH (10 mM). The concentration of aPD-L1 was then determined using the ELISA protocol designed above.
  • Alexa 647-labeled aPD-L1 and Alexa 647-labeled Gluc-S-aPD-L1 (1.0 mg kg ⁇ 1 ) with varying glucose densities were administered to C57BL/L1 with orthotopic GL261 tumors.
  • 6J mice were dosed intravenously and sacrificed 1, 4, 12 and 24 hours later for heart, liver, spleen, lung, kidney, brain and tumor extraction. Next, each tissue was washed with D-PBS(-), excess washing solution was removed, weighed, and homogenized with 600 ⁇ L of cell lysis buffer. Finally, the biodistribution of aPD-L1 and Gluc-S-aPD-L1 was quantified by fluorescence measurements using an Infinite M1000 PRO spectrophotometer (Tecan Group Ltd., Mannedorf, Switzerland).
  • various formulations 1.5 mg kg ⁇ 1 for aPD-L1
  • Bioluminescence signals from GL261-luc or CT2A-luc tumors were observed with an in vivo imaging system (IVIS) after injection of 150 mg kg ⁇ 1 of luciferin. Mouse survival time was followed and the significance of the prolongation was determined by log-rank test.
  • IVIS in vivo imaging system
  • C57BL/6J mice were first injected with 1.0 ⁇ 10 5 GL261 cells followed by Gluc25-S-aPD-L1. Injections were made 90 days after treatment. Bioluminescence signals were then monitored and survival time recorded.
  • IFN ⁇ ELISA kit BioLegend; Catalog No. 430804
  • Peripheral blood was collected 3 days after injection of aPD-L1, Gluc0-S-aPD-L1 and Gluc25-S-aPD-L1 and centrifuged at 500 g for 10 minutes. Supernatants were aliquoted and stored at -80°C until analysis. Samples were diluted in ELISA assay buffer according to the manufacturer's instructions and analyzed using an ELISA kit.
  • tumor tissue was excised 3 days after injection of aPD-L1, Gluc0-S-aPD-L1 and Gluc25-S-aPD-L1 and dissociated with BD Horizon TM Dri Tissue & Tumor Dissociation Reagent. let me Tumor cells were then stained with anti-CD3, anti-CD8, anti-CD4, anti-CD45, or anti-Foxp3 antibodies for 30 minutes. Foxp3 staining was performed according to Biolegend's intracellular staining protocol. Stained cells were then subjected to flow cytometry for analysis of CD8 and Foxp3 cell populations (2 ⁇ 10 4 cells were collected for analysis).
  • splenocytes were harvested from saline and Gluc25-S-aPD-L1 treated mice after 60 days and stained with anti-CD8, anti-CD44 and anti-CD62L antibodies. Stained cells were then analyzed by flow cytometry (2 ⁇ 10 4 cells were collected for analysis).
  • TNF- ⁇ , IL-6 and IL-1 ⁇ levels in each tissue were measured using ELISA kits (Thermo Fisher, catalog numbers BMS607-3, KMC0061 and BMS6002).
  • Tissues including lung, liver, and kidney were treated according to instructions, then homogenized and centrifuged at 500 g for 10 minutes. Supernatants were aliquoted and stored at -80°C until analysis. Samples were diluted in ELISA assay buffer according to the manufacturer's instructions and analyzed using an ELISA kit. Alexa647-labeled aPD-L1 and Alexa647-labeled Gluc25-S-aPD-L1 were administered intravenously to mice and sacrificed 24 hours later to extract lungs, livers and kidneys.
  • Tissues were then cut into 10.0 ⁇ m sections, which were fixed with cold acetone and washed with PBS. It was then blocked with 5% BSA for 1 hour at room temperature. Sections were then incubated with Alexa488-labeled anti-human IgG (H+L) (Thermo Fisher Scientific, Catalog No. A-11013) overnight at 4° C., followed by CLSM observation.
  • H+L Alexa488-labeled anti-human IgG
  • a characteristic peak of the polymer was identified in the 1 H nuclear magnetic resonance (NMR) spectrum.
  • NMR nuclear magnetic resonance
  • a series of Gluc-S-NPCs with different glucose concentrations (0, 25, 50, 100 mol%) were prepared, Then, it was reacted with the amino group of aPD-L1 by a covalent bonding method. After that, when the residue amino groups of aPD-L1 were detected using fluorescamine as a probe, it was found that about 60% of the amino groups were modified (Fig. 3).
  • DLS dynamic light scattering
  • aPD-L1 had a zeta potential of ⁇ 6.13 mV and Gluc-S-aPD-L1 had a zeta potential of ⁇ 0.16 mV, whereas glutathione (GSH) treatment resulted in a reduction of PEG chains from the antibody. Elimination occurred, resulting in a negative charge of ⁇ 6.07 mV, which is almost the same as the zeta potential of unmodified aPD-L1 (Fig. 4 right panel). Thus, as shown in FIG. 5, aPD-L1 increases in size and zeta- potential rises.
  • Gluc-S-aPD-L1 reverts to the unmodified form of aPD-L1 by cleaving the disulfide bond in the PEG chain under a reducing environment (eg, in the presence of GSH).
  • GPC gel permeation chromatography
  • Gluc-S-aPD-L1 showed no significant difference in ⁇ -sheet absorbance compared to unmodified aPD-L1, suggesting that Gluc-S-aPD-L1 with well-preserved structure It was suggested that L1 maintains physiological function to PD-L1 after PEG chain cleavage 25 . Except for the secondary structure, the surface structure of aPD-L1, which does not contain residues occupying the amine groups of lysines, is preserved after responsive PEG chain cleavage, thus maintaining physiological function. was further confirmed (Fig. 5).
  • an enzyme-linked immunosorbent assay (ELISA) 26 was used to quantify the specific binding ability of Gluc-S-aPD-L1 to its ligands. . As shown in FIG. 6, right, Gluc-S-aPD-L1 did not show significant binding to PD-L1 in ELISA. In contrast, when Gluc-S-aPD-L1 was exposed to a reducing environment (1.0 mM GSH, pH 7.4), the antibody binding amount increased significantly in a time-dependent manner (2 hours or longer).
  • PD-L1 is primarily expressed on the membrane of tumor cells28 .
  • the specific binding of Gluc-S-aPD-L1 to GL261 glioblastoma cells was assessed.
  • Unmodified aPD-L1 as a positive control showed favorable co-localization with tumor cell membranes after 1.0 hour incubation, but negligible after incubation of Gluc-S-aPD-L1 with tumor cells. Only moderate red fluorescence was observed (Fig. 7). This is probably because Gluc-S-aPD-L1 significantly impairs the binding of the antibody to PD-L1 due to modification with a PEG chain.
  • the brain parenchyma is known to be a reducing environment, and Gluc-S-aPD-L1 restores binding affinity specifically in the brain parenchyma, and is effective for site-specific ICB therapy in the brain (especially in other organs). antibody is inactive).
  • GBM blood-brain barrier
  • aPD-L1 was first labeled with a fluorescent dye and then modified with Gluc-PEG-S-NPC and PEG-S-NPC, respectively. Unmodified aPD-L1 and Gluc-S-aPD-L1 with varying modified glucose densities were injected i.v. Removed for fluorescence analysis during 24 hours post-injection. Clearly, unmodified aPD-L1 and Gluc0-S-aPD-L1, which does not have active targeting ability as a control, accumulated in tumors in the brain during 24 hours, but the amount was small.
  • Gluc25-S-aPD-L1 showed the highest accumulation with an ID g- 1 of 2.2%, ⁇ 6.1-fold higher than the Gluc0-S-aPD-L1 group in the first 4 hours, and unmodified aPD-L1 It showed an increased amount of accumulation of ⁇ 18.0-fold over the L1 group (Fig. 9 left panel). This indicates that the antibody enhanced active transport across the BBB with modified glucose 31-33 .
  • Gluc-S-aPD-L1 is also accumulated in the liver, spleen, kidney, and lungs, and aPD-L1 introduced with high glucose density is easily taken up by the liver, and Gluc50-S-aPD-L1 and It correlates with decreased blood circulation of Gluc100-S-aPD-L1.
  • the technology of the present invention is effective in immune checkpoint inhibitor (ICB) therapy in the brain.
  • IDB immune checkpoint inhibitor
  • no special blood sugar manipulation particularly, blood sugar manipulation such as administering an antibody together with a blood sugar level increasing manipulation after fasting
  • the BBB is disrupted in subjects with brain tumors, allowing drugs to reach the brain tumor.
  • the GL261 cell line was inoculated intracranially on the right side of the brain.
  • GL261 tumor-bearing mice were then given a single dose of saline, aPD-L1, Gluc0-S-aPD-L1, Gluc25-S-aPD-L1 at a dose of 1.5 mg/kg.
  • inactivated (uncleaved) Gluc25-C-aPD-L1 was applied as a control.
  • Brain tumor development was followed by bioluminescence of GL261 cells in the whole brain.
  • mice treated with Gluc25-S-aPD-L1 had reduced bioluminescence after 6 days, with 2 out of 5 mice having negligible levels of GL261 cells in brain regions. was detected, suggesting a strong immune response.
  • mice receiving unmodified aPD-L1 and Gluc0-S-aPD-L1 had only weak therapeutic effects against GL261 tumors.
  • the therapeutic effect was dependent on the amount of antibody accumulated in the brain.
  • GL261 tumors from mice treated with inactivated (uncleaved) Gluc25-C-aPD-L1 also showed a robust growth profile, which was consistent with inactivated (uncleaved) Gluc25-C-aPD-L1. This is due to the fact that aPD-L1 does not release aPD-L1 in response to reduction, and the presence of a reducing environment and a linker responsive to the reducing environment plays an important role in the brain-directed ICB therapy of the present invention. (Fig.
  • mice treated with Gluc25-S-aPD-L1 had a survival rate of approximately 60% after 90 days, but no mice survived more than 32 days, and all other control groups showed significant weight loss. (Fig. 10-2).
  • Gluc25-S-aPD-L1 Given the potent anti-cancer effect on GL261, we also applied Gluc25-S-aPD-L1 to treat the CT-2A cell line.
  • the CT-2A cell line exhibits low levels of PTEN expression34, accurately reflecting several features of clinical glioblastoma , including intratumoral heterogeneity, and a high proportion of cancers. Due to the presence of stem cells, it is radio- and chemo-resistant 34,35 .
  • CT-2A tumor-bearing mice in the saline-treated group showed aggressive tumor growth with a survival time of less than 20 days (FIG. 11). The same was true for the unmodified aPD-L1 administration group. In contrast, mice treated with Gluc25-S-aPD-L1 significantly retarded tumor growth and prolonged survival (FIG. 11). This suggests that both aggressive delivery of ICB antibodies via the BBB and reactivatable ICB therapy can be therapeutically effective against highly aggressive glioblastoma through a robust and robust anti-tumor immune response. increase the
  • aPD-L1 was first labeled with Alexa647 dye, then Gluc0-S-aPD-L1, Gluc25-S-aPD-L1 and Gluc25-C-aPD-L1 were prepared respectively.
  • Various formulations were then injected intravenously into mice bearing orthotopic brain tumors. Twenty-four hours later, mice were sacrificed and tumor tissue was removed. Tissue sections were made from the removed tissue and incubated with anti-human secondary antibody. As shown in FIG.
  • mice treated with Gluc25-C-aPD-L1 showed negligible Alexa488 fluorescence due to difficulty in PEG chain cleavage (FIG. 13, lower right panel). Therefore, the reductive environment-dependent aPD-L1 release behavior of Gluc-S-aPD-L1 via PEG chain scission is expected to reduce the side effects of the inactivated antibody outside the brain tissue and reactivate it in the brain. This will open the way for ICB therapy based on biochemistry.
  • mice treated with Gluc25-S-aPD-L1 splenic T cells were harvested and analyzed using flow cytometry. did.
  • the CD44hiCD62Llow effector memory T cell subset increased approximately 2.0-fold compared to native mice (Fig. 16).
  • the function of effector memory T cells was further confirmed by tumor rechallenge experiments. Mice in which Gluc25-S-aPD-L1 was administered on the right side of the brain to cause tumor disappearance were re-challenged with GL261 tumor cells on the left side of the brain (FIG. 17-1), as shown in FIG. 17-2.
  • aPD-L1 PEGylation strategy was effective in enhancing the delivery of antibodies to the vascular endothelial wall. It suggests that it effectively blocked the binding affinity (Fig. 8 right panel). Additionally, to demonstrate reduced off-target effects, select non-target tissues (lung, liver, kidney, etc.) to suppress immune-related adverse events (irAEs) using Gluc25-S-aPD-L1 evaluated the possibilities.
  • Adverse events resulting from off-target effects are widely predicted to be hyperactivation of the immune system within the parenchyma of non-target tissues, accompanied by lymphocyte infiltration and production of inflammatory cytokines 11-13 .
  • Five days post-injection of unmodified aPD-L1 and Gluc25-S-aPD-L1, lungs and kidneys were enriched with infiltrating lymphocytes (CD45+ cells) and pro-inflammatory cytokines (e.g., TNF- ⁇ , IL-6 and IL-1 ⁇ ) were excised for measurement of release. As shown in FIG.
  • mice in the Gluc25-S-aPD-L1-administered group showed relatively low lymphocyte infiltration, almost equivalent to the saline-administered group, indicating that the non-activating function of Gluc25-S-aPD-L1 was non-existent. It was suggested that it contributes to the reduction of the risk of excessive infiltration of lymphocytes in the target tissue.
  • aPD-L1 was significantly more active than saline-treated or Gluc25-S-aPD-L1-treated groups. It was found that TNF- ⁇ , IL-6 and IL-1 ⁇ levels in 25% of the mice treated with 2- to 4-fold abnormally increased (Fig. 19). In addition, tissue sections were stained to investigate whether aPD-L1 could be released from Gluc25-S-aPD-L1 in non-target tissues (FIG. 20).
  • the GLUT1 ligand was linked to PEG as a brain-targeting molecule in order to efficiently deliver antibodies to the brain.
  • PEG-modified antibodies without targeting molecules were generated for extrabrain antibody delivery and cancer therapy experiments.
  • the antibody used below is a modified antibody in which an uncharged hydrophilic polymer block, a reducing environment-responsive linker and an antibody are linked in that order.
  • PEG was used as the non-charged hydrophilic polymer block in the same manner as described above. PEGylation caused the antibody to lose antigen-binding activity.
  • Tail vein injection of physiological saline, unmodified anti-PD-L1 antibody, and PEG-modified anti-PD-L1 antibody without targeting molecule to subcutaneous transplantation mouse model of malignant melanoma cell line (B16-F10) (n 5 in each experimental group).
  • a PEG-modified anti-PD-L1 antibody was produced in the same manner as the antibody produced in the above example. The dose was adjusted so that the amount of antibody was 1.5 mg/kg body weight. Dosing was performed three times every other day. The tumor volume (V) was measured on the first day of administration and every three days, and the ratio (V/V0) of the tumor volume (V) to the tumor volume (V0) on the first day was determined.
  • the inside of the tumor tissue is generally a reducing environment.
  • Tumors generally have a hypoxic environment due to lack of nutrients and oxygen supply for cell growth. Even in a hypoxic environment, tumor tissue becomes a reductive environment due to reductase abundantly present in cancer cells.
  • a PEG-modified antibody was designed to cleave the PEG and revert to a native antibody responsive to a reducing environment. This antibody was found to restore the lost antigen-binding activity in response to the reducing environment within the tumor in vivo, and to exert similar effects to the unmodified antibody.
  • the PEG-modified antibody of the present invention can reduce side effects outside of tumor tissue and/or increase the amount delivered to tumor tissue by eliminating its binding activity through modification. may achieve reduced side effects and/or improved delivery to tumor tissue than conventional unmodified antibodies.
  • Kadcyla (Roche) was used as an ADC.
  • Kadcyla is an ADC represented by the following formula (I) obtained by linking trastuzumab and emtansine via a linker (hereinafter also referred to as "T-DM1"), and is used as an anticancer agent.
  • the above ADC was modified with PEG to obtain a modified ADC.
  • the PEG modification is a cleavable linker- PEG was used.
  • R was a methoxy group.
  • R can also be glucose or a reactive group such as an azide group.
  • Reactive groups can incorporate targeting molecules for targeting to tissues within the body.
  • This linker is linked to some amino groups abundantly present in antibodies, and under a reducing environment, it is cleaved from the linker to release the ADC (unmodified ADC) from the modified ADC.
  • ADC unmodified ADC
  • PEG modification of ADC and release of ADC (unmodified ADC) under reducing environment were tested.
  • Kadcyla 4.10 mg/ml (T-DM1 1.0 mg/ml) and PEG-(CH 2 ) 2 -SS-(CH 2 ) 2 - dissolved in 20 mM phosphate buffer (pH 7.4) NPC 10 mg/ml was mixed 1:1 (volume ratio) and allowed to react overnight at room temperature.
  • the reaction solution was purified by ultrafiltration (Vivaspin 6, MWCO: 30 kDa, 4000 g, 4° C.) five times and processed with a 0.1 ⁇ m PVDF filter to obtain PEG-modified T-DM1.
  • Particle size distribution and scattered light intensity were measured with a Zetasizer Nano ZS (532 nm, 173°).
  • T-DM1 not modified with PEG was measured.
  • the results were as shown in FIG.
  • PEG-modified T-DM1 PEG-modified T-DM1
  • PEG-modified antibodies that retain their structural integrity in non-target tissues (e.g., lung, liver, kidney) have their antigen specificity blocked so as not to induce the development of immune-related adverse events. Designed.
  • PEGylation process has been demonstrated in several FDA-approved drugs such as Doxil 23.
  • Linking PEG chains to polypeptides, other candidate molecules or nanocarriers can lead to rapid elimination, pseudo-allergic reactions. , and poor target accumulation are long-standing strategies to overcome drug deficiencies.
  • the present strategy using PEGylated antibodies could serve as a general and versatile platform for rationally designed protein delivery systems.
  • reactive properties such as acidic pH, oxidative stress, and overexpressed enzymes within the TME can be site-specifically activated by appropriate chemical design 40 .
  • Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756-760 (2006).

Abstract

The present invention relates to a technique for delivering an antibody to the brain or a tumor tissue. The present invention provides a modified antibody that is targeted. The present invention provides an inactivated antibody that is targeted, the inactivated antibody being a modified antibody that can be re-activated in the brain or a tumor tissue.

Description

抗原への結合親和性を低減させた抗体Antibodies with reduced binding affinity to antigen
　本発明は、抗原への結合親和性を低減させた抗体に関する。 The present invention relates to antibodies with reduced binding affinity to antigens.
　抗体医薬が世界中で疾患の治療のために開発されている。抗体は、分子標的薬であり、それ故に理想的には標的組織に特異的である。しかしながら、実際には、抗体の抗原は標的組織以外にも発現があるために、完全に標的組織特異的なものとはならないケースが多い。特に、がん抗原を標的化した抗体を含む抗がん剤において、標的組織特異性が低い場合には、腫瘍以外を損傷し、副作用が生じ得る。 Antibody drugs are being developed for the treatment of diseases around the world. Antibodies are molecularly targeted drugs and are therefore ideally specific to the target tissue. In practice, however, antibody antigens are expressed in tissues other than target tissues, and therefore are not completely target tissue-specific in many cases. In particular, anticancer agents containing antibodies targeting cancer antigens may damage areas other than tumors and cause side effects if the target tissue specificity is low.
　最も進行性の高い脳腫瘍である膠芽腫（ＧＢＭ）は、死亡率が最も高く、生存期間が短く、予後不良であることが特徴であり、普遍的に致死的である（非特許文献１～３）。モノクローナル抗体を用いた免疫チェックポイント阻害薬（ＩＣＢ）療法は、がん治療に革命をもたらした（非特許文献４～６）。しかし、血液脳関門（ＢＢＢ）を越えた抗体の送達には限界があり、膠芽腫（ＧＢＭ）患者ではＩＣＢ治療に反応することはまれである（非特許文献７～９）。また、ＩＣＢ療法により自己免疫活性化、リンパ球浸潤、炎症性サイトカインの放出などの免疫関連有害事象（ｉｒＡＥ）を生じることがある（非特許文献１０～１３）。ＩＣＢ治療では、血管内皮細胞、筋肉、肝細胞、膵島細胞でＰＤ－Ｌ１が発現しているため、正常組織へのオフターゲット結合が常に発生しており、したがって、抗ＰＤ－Ｌ１抗体（ａＰＤ－Ｌ１）を全身投与した場合には、ｉｒＡＥｓの発生率が増加し、治療効果が低下する（非特許文献１４～１６）。 Glioblastoma (GBM), the most aggressive brain tumor, is characterized by the highest mortality, short survival, and poor prognosis, and is universally fatal (Non-Patent Document 1- 3). Immune checkpoint inhibitor (ICB) therapy using monoclonal antibodies has revolutionized cancer therapy (Non-Patent Documents 4-6). However, delivery of antibodies across the blood-brain barrier (BBB) is limited, and glioblastoma (GBM) patients rarely respond to ICB treatment (7-9). In addition, ICB therapy may cause immune-related adverse events (irAE) such as autoimmune activation, lymphocyte infiltration, and release of inflammatory cytokines (Non-Patent Documents 10-13). In ICB treatment, PD-L1 is expressed in vascular endothelial cells, muscle, hepatocytes, and pancreatic islet cells, so that off-target binding to normal tissues always occurs, thus anti-PD-L1 antibodies (aPD- When L1) is administered systemically, the incidence of irAEs increases and the therapeutic effect decreases (Non-Patent Documents 14-16).
ＷＯ２０１２／１１２６８９AWO2012/112689A
　本発明によれば、抗原への結合親和性を低減させた抗体が提供される。本発明によればまた、環境応答性に再活性化する抗体が提供される。 According to the present invention, antibodies with reduced binding affinity to antigens are provided. The present invention also provides antibodies that reactivate in environmental responsiveness.
　本発明者らは、抗体を非電荷親水性ポリマーで修飾し、抗体を覆うことによって、抗体の抗原に対する親和性を低下させることで、体内において抗体を非標的組織では不活性化状態（抗原への結合親和性が減弱または喪失した状態）とすることができ、および、非電荷親水性ポリマーによる修飾を標的組織内で開裂するようにリンカーを設計し、標的組織内で抗体からの修飾が除去されるようにすることで、体内において標的組織においてのみ抗体を活性化状態（抗原への親和性がより高まった状態）にすることができることを見出した。 The present inventors have found that by modifying an antibody with a non-charged hydrophilic polymer and covering the antibody to reduce the affinity of the antibody for the antigen, the antibody is inactivated in non-target tissues in the body (to the antigen). with reduced or lost binding affinity), and the linker is designed to cleave the modification with the uncharged hydrophilic polymer within the target tissue, removing the modification from the antibody within the target tissue. The inventors have found that by allowing the antibody to be activated, it is possible to activate the antibody (a state in which the affinity for the antigen is higher) only in the target tissue in the body.
　本発明によれば、以下の発明が提供される。
［１］非電荷親水性ポリマーブロック（好ましくは、ポリエチレングリコール（ＰＥＧ））により修飾された修飾抗体であって、非電荷親水性ポリマーブロック、環境応答性結合、および抗体が、この順番で連結し、各連結は、スペーサーを介していてもよく、環境応答性結合は、還元環境下で開裂する結合である、修飾抗体｛ここで、特に限定されないが、抗体は、好ましくは、免疫チェックポイント分子に結合して免疫チェックポイントを阻害する｝。
［２］その抗原に対する結合親和性（ＫＤ）が、前記修飾前（未修飾型抗体）と比較して、１０％以下である、上記［１］に記載の抗体。
［３］その抗原に対する結合親和性（ＫＤ）が、前記修飾前（未修飾型抗体）と比較して、５％以下である、上記［１］に記載の抗体。
［４］血清環境下において、その抗原に対して実質的にまたは有意に結合しない｛例えば、結合が検出限界以下である｝、上記［１］に記載の抗体。
［５］環境応答性結合は、脳実質における還元環境下、または腫瘍組織における還元環境下において、開裂する結合である、上記［１］～［４］のいずれか一項に記載の抗体。
［６］環境応答性結合が開裂すると、その抗原に対する結合親和性（ＫＤ）が、回復する、上記［５］に記載の抗体。
［７］－Ｃ（Ｏ）－Ｏ－Ｌ1－Ｓ－Ｓ－Ｌ2－（非電荷親水性ポリマーブロック）により修飾されたアミノ基を有する、上記［１］～［６］のいずれかに記載の抗体
｛ここで、
Ｌ1は置換されていてもよい低級アルキレンであり、
Ｌ2は結合または生体内において安定なリンカーである｝。
［８］Ｌ1が、エチレンである、上記［７］に記載の抗体。
［９］ＰＤ－１系免疫チェックポイントを阻害する、上記［１］～［８］のいずれかに記載の抗体。
［１０］ＰＤ－Ｌ１またはＰＤ－１に結合する抗体である、上記［９］に記載の抗体。
［１１］標的化分子を表出している、上記［１］～［１０］のいずれかに記載の抗体。
［１２］標的化分子が、非電荷親水性ポリマーブロックと連結している、上記［１１］に記載の抗体。
［１３］標的化分子が、ＧＬＵＴ１リガンドである、上記［１２］に記載の抗体。
［１４］ＧＬＵＴ１リガンドが、グルコースである、上記［１３］に記載の抗体。
［１５］免疫チェックポイント分子に結合して、免疫チェックポイント分子間の相互作用を中和する抗体である、上記［１］～［１４］のいずれかに記載の抗体。
［１６］免疫チェックポイント分子が、免疫細胞に発現する免疫チェックポイント分子のカウンターパートである、上記［１５］に記載の抗体。
［１７］免疫チェックポイント分子が、免疫細胞に発現する免疫チェックポイント分子である、上記［１５］に記載の抗体。
［１８］上記［１］～［１７］のいずれかに記載の抗体を含む、医薬組成物。
［１９］抗体のアミノ酸残基のアミノ基が、非電荷親水性ポリマーブロックおよび環境応答性結合と連結している、上記［１］～［６］のいずれかに記載の抗体。
［２０］上記［１８］に記載の抗体を含む、医薬組成物。
［２１］抗原が、腫瘍抗原である、上記［１］～［１７］および［１９］のいずれかに記載の抗体。
［２２］腫瘍抗原が、非脳腫瘍の抗原である、上記［２１］に記載の抗体。
［２３］腫瘍抗原が、脳腫瘍の抗原である、上記［２２］に記載の抗体。
［２４]標的腫瘍に対する標的化分子を表出する、上記［１］～［１７］および［１９］～［２３］のいずれかに記載の抗体。
［２５］標的抗原が、非電荷親水性ポリマーブロックに連結している、上記［２４］に記載の抗体。
［２６］上記［１］～［１７］および［１９］～［２５］のいずれかに記載の抗体を含む、医薬組成物。
［２７］がんを処置することに用いるための、上記［２６］に記載の医薬組成物。
［２８］上記いずれかに記載の抗体の抗原結合性断片。
［２９］上記［２８］の抗原結合性断片を含む医薬組成物。
［３０］抗体が、薬物抗体コンジュゲート（ＡＤＣ）の形態である、上記いずれかに記載の抗体。
［３１］平均流体力学径（体積平均）が、６ｎｍ～１２ｎｍ（１２ｎｍまたは１２ｎｍ未満）である、上記［３０］に記載の抗体。
［３２］未修飾型ＡＤＣの平均流体力学径（体積平均）が、１２ｎｍ以下である、上記［３１］に記載の抗体。
［３３］ＡＤＣが、抗体部分が有する側鎖アミノ基に対して－リンカー－薬物が導入されたものである、上記［３０］～［３２］のいずれかに記載の抗体。
［３４］ＡＤＣが、抗体部分が有する側鎖アミノ基に対して２～４個の－リンカー－薬物が導入されたものである、上記［３０］～［３３］に記載の抗体。すなわち、ＡＤＣは、抗体部分－（リンカー－薬物）ｎ｛ここで、ｎは、数平均であり、２～１０である。｝で示される。
［３５］ＡＤＣが、抗体部分が有する側鎖アミノ基に対して２～４個の－リンカー－薬物が導入されたものであり、平均流体力学径（体積平均）が、１２ｎｍ～３０ｎｍ（特に、１３ｎｍ～２５ｎｍ）である、上記［３０］に記載の抗体。
［３６］非修飾型ＡＤＣの平均流体力学径（体積平均）が、１２ｎｍ以下である、上記［３５］に記載の抗体。
［３７］抗体部分が結合する抗原が、がん抗原である、上記いずれかに記載の抗体。
［３８］上記いずれかに記載の抗体を含む、組成物。
［３９］上記いずれかに記載の抗体を含む、抗体部分が結合する抗原に対する体内における標的化に用いるための組成物。
［４０］ＡＤＣが、ゲムツズマブ オゾガマイシン（マイロターグ）、イブリツモマブ チウキセタン（ゼヴァリン）、ブレンツキシマブ ベドチン（アドセトリス）、トラスツズマブ エムタンシン（カドサイラ）、イノツズマブ オゾガマイシン（ベスポンサ）、モキセツモマブ パスドトックス－ｔｄｆｋ（LUMOXITI）、ポラツズマブ ベドチン－Ｐｉｉｑ（Polivy）、トラスツズマブ デルクステカン（エンハーツ）、およびエンホルツマブ ベドチン（PADCEV）からなる群から選択される１以上を含む、上記いずれかに記載の抗体。
［４１］上記［４０］に記載の抗体を含む、組成物。
［４２］がんを処置することに用いるための、上記［４１］に記載の組成物。 According to the present invention, the following inventions are provided.
[1] A modified antibody modified with an uncharged hydrophilic polymer block (preferably polyethylene glycol (PEG)), wherein the uncharged hydrophilic polymer block, the environment-responsive linkage, and the antibody are linked in that order. , each linkage may be via a spacer, the environmentally responsive bond is a bond that cleaves under a reducing environment, the modified antibody {here, although not particularly limited, the antibody is preferably an immune checkpoint molecule binds to and inhibits immune checkpoints}.
[2] The antibody of [1] above, whose binding affinity (KD) for the antigen is 10% or less compared to the unmodified antibody (unmodified antibody).
[3] The antibody of [1] above, whose binding affinity (KD) for the antigen is 5% or less compared to the unmodified antibody (unmodified antibody).
[4] The antibody of [1] above, which does not substantially or significantly bind to its antigen in a serum environment {eg, binding is below the detection limit}.
[5] The antibody according to any one of [1] to [4] above, wherein the environment-responsive bond is a bond that cleaves under a reducing environment in brain parenchyma or in a reducing environment in tumor tissue.
[6] The antibody of [5] above, wherein the binding affinity (KD) for the antigen is restored when the environment-responsive bond is cleaved.
[7] Any of the above [1] to [6] having an amino group modified with —C(O)—OL 1 —S—S—L 2 — (uncharged hydrophilic polymer block) The antibody described {wherein
L 1 is optionally substituted lower alkylene,
L2 is a bond or an in vivo stable linker } .
[8] The antibody of [7] above, wherein L 1 is ethylene.
[9] The antibody of any one of [1] to [8] above, which inhibits the PD-1 system immune checkpoint.
[10] The antibody of [9] above, which is an antibody that binds to PD-L1 or PD-1.
[11] The antibody according to any one of [1] to [10] above, which expresses a targeting molecule.
[12] The antibody of [11] above, wherein the targeting molecule is linked to an uncharged hydrophilic polymer block.
[13] The antibody of [12] above, wherein the targeting molecule is a GLUT1 ligand.
[14] The antibody of [13] above, wherein the GLUT1 ligand is glucose.
[15] The antibody according to any one of [1] to [14] above, which binds to an immune checkpoint molecule and neutralizes the interaction between the immune checkpoint molecules.
[16] The antibody of [15] above, wherein the immune checkpoint molecule is a counterpart of an immune checkpoint molecule expressed on immune cells.
[17] The antibody of [15] above, wherein the immune checkpoint molecule is an immune checkpoint molecule expressed in immune cells.
[18] A pharmaceutical composition comprising the antibody of any one of [1] to [17] above.
[19] The antibody according to any one of [1] to [6] above, wherein the amino group of the amino acid residue of the antibody is linked to the uncharged hydrophilic polymer block and the environment-responsive bond.
[20] A pharmaceutical composition comprising the antibody of [18] above.
[21] The antibody of any one of [1] to [17] and [19] above, wherein the antigen is a tumor antigen.
[22] The antibody of [21] above, wherein the tumor antigen is a non-brain tumor antigen.
[23] The antibody of [22] above, wherein the tumor antigen is a brain tumor antigen.
[24] The antibody of any one of [1]-[17] and [19]-[23] above, which displays a targeting molecule against a target tumor.
[25] The antibody of [24] above, wherein the target antigen is linked to an uncharged hydrophilic polymer block.
[26] A pharmaceutical composition comprising the antibody of any one of [1] to [17] and [19] to [25] above.
[27] The pharmaceutical composition of [26] above for use in treating cancer.
[28] An antigen-binding fragment of the antibody according to any one of the above.
[29] A pharmaceutical composition comprising the antigen-binding fragment of [28] above.
[30] The antibody of any of the above, wherein the antibody is in the form of a drug-antibody conjugate (ADC).
[31] The antibody of [30] above, which has an average hydrodynamic diameter (volume average) of 6 nm to 12 nm (12 nm or less than 12 nm).
[32] The antibody of [31] above, wherein the unmodified ADC has an average hydrodynamic diameter (volume average) of 12 nm or less.
[33] The antibody according to any one of [30] to [32] above, wherein the ADC has a -linker-drug introduced to the side chain amino group of the antibody portion.
[34] The antibody according to [30] to [33] above, wherein 2 to 4 -linker-drugs are introduced to the side chain amino groups of the antibody portion. That is, the ADC is antibody moiety-(linker-drug) n {where n is the number average and ranges from 2-10. }.
[35] The ADC has 2 to 4 -linker-drugs introduced to the side chain amino groups of the antibody moiety, and has an average hydrodynamic diameter (volume average) of 12 nm to 30 nm (especially 13 nm to 25 nm), the antibody according to [30] above.
[36] The antibody of [35] above, wherein the unmodified ADC has an average hydrodynamic diameter (volume average) of 12 nm or less.
[37] The antibody of any of the above, wherein the antigen to which the antibody portion binds is a cancer antigen.
[38] A composition comprising the antibody of any of the above.
[39] A composition for use in vivo targeting of an antigen to which the antibody portion binds, comprising the antibody of any of the above.
[40] ADC is gemtuzumab ozogamicin (Milotarg), ibritumomab tiuxetan (Zevalin), brentuximab vedotin (ADCETRIS), trastuzumab emtansine (Kadcyra), inotuzumab ozogamicin (Vesponsa), moxetumomab pasudotox-tdfk (LUMOXITI), polatuzumab iq vedotin (Polivy), trastuzumab deruxtecan (Enherts), and enfortumab vedotin (PADCEV).
[41] A composition comprising the antibody of [40] above.
[42] The composition of [41] above for use in treating cancer.
［１］免疫チェックポイント分子に結合して免疫チェックポイントを阻害する抗体であって、非電荷親水性ポリマーブロック（好ましくは、ポリエチレングリコール（ＰＥＧ）により修飾されており、非電荷親水性ポリマーブロック、環境応答性結合、および抗体が、この順番で連結し、各連結は、スペーサーを介していてもよく、環境応答性結合は、還元環境下で開裂する結合である、抗体（例えば、修飾抗体）。
［２］その抗原に対する結合親和性（ＫＤ）が、前記修飾前と比較して、１０％以下である、上記［１］に記載の抗体。
［３］その抗原に対する結合親和性（ＫＤ）が、前記修飾前と比較して、５％以下である、上記［１］に記載の抗体。
［４］血清環境下において、その抗原に対して実質的にまたは有意に結合しない｛例えば、結合が検出限界以下である｝、上記［１］に記載の抗体。
［５］環境応答性結合は、脳実質における還元環境下、または腫瘍組織における還元環境下において、開裂する結合である、上記［１］～［４］のいずれか一項に記載の抗体。
［６］環境応答性結合が開裂すると、その抗原に対する結合親和性（ＫＤ）が、回復する、上記［５］に記載の抗体。
［７］－Ｃ（Ｏ）－Ｏ－Ｌ1－Ｓ－Ｓ－Ｌ2－（非電荷親水性ポリマーブロック）により修飾されたアミノ基を有する、上記［１］～［６］のいずれかに記載の抗体
｛ここで、
Ｌ1は置換されていてもよい低級アルキレンであり、
Ｌ2は結合または生体内において安定なリンカーである｝。
［８］Ｌ1が、エチレンである、上記［７］に記載の抗体。
［９］ＰＤ－１系免疫チェックポイントを阻害する、上記［１］～［８］のいずれかに記載の抗体。
［１０］ＰＤ－Ｌ１またはＰＤ－１に結合する抗体である、上記［９］に記載の抗体。
［１１］標的化分子を表出している、上記［１］～［１０］のいずれかに記載の抗体。
［１２］標的化分子が、非電荷親水性ポリマーブロックと連結している、上記［１１］に記載の抗体。
［１３］標的化分子が、ＧＬＵＴ１リガンドである、上記［１２］に記載の抗体。
［１４］ＧＬＵＴ１リガンドが、グルコースである、上記［１３］に記載の抗体。
［１５］免疫チェックポイント分子に結合して免疫チェックポイントを阻害する抗体は、免疫チェックポイント分子に結合して、免疫チェックポイント分子間の相互作用を中和する抗体である、上記［１］～［１４］のいずれかに記載の抗体。
［１６］免疫チェックポイント分子が、免疫細胞に発現する免疫チェックポイント分子のカウンターパートである、上記［１５］に記載の抗体。
［１７］免疫チェックポイント分子が、免疫細胞に発現する免疫チェックポイント分子である、上記［１５］に記載の抗体。
［１８］上記［１］～［１７］のいずれかに記載の抗体を含む、医薬組成物。
［１９］抗体のアミノ酸残基のアミノ基が、非電荷親水性ポリマーブロックおよび環境応答性結合と連結している、上記上記［１］～［６］のいずれかに記載の抗体。
［２０］上記［１８］に記載の抗体を含む、医薬組成物。
［２１］がんを処置することに用いるための、上記［２０］に記載の医薬組成物。 [1] An antibody that binds to an immune checkpoint molecule and inhibits the immune checkpoint, which is modified with an uncharged hydrophilic polymer block (preferably polyethylene glycol (PEG)), An antibody (e.g., a modified antibody) in which an environmentally responsive bond and an antibody are linked in that order, each linkage may be via a spacer, and the environmentally responsive bond is a bond that is cleaved under a reducing environment .
[2] The antibody of [1] above, whose binding affinity (KD) for the antigen is 10% or less compared to before the modification.
[3] The antibody of [1] above, whose binding affinity (KD) for the antigen is 5% or less compared to before the modification.
[4] The antibody of [1] above, which does not substantially or significantly bind to its antigen in a serum environment {eg, binding is below the detection limit}.
[5] The antibody according to any one of [1] to [4] above, wherein the environment-responsive bond is a bond that cleaves under a reducing environment in brain parenchyma or in a reducing environment in tumor tissue.
[6] The antibody of [5] above, wherein the binding affinity (KD) for the antigen is restored when the environment-responsive bond is cleaved.
[7] Any of the above [1] to [6] having an amino group modified with —C(O)—OL 1 —S—S—L 2 — (uncharged hydrophilic polymer block) The antibody described {wherein
L 1 is optionally substituted lower alkylene,
L2 is a bond or an in vivo stable linker } .
[8] The antibody of [7] above, wherein L 1 is ethylene.
[9] The antibody of any one of [1] to [8] above, which inhibits the PD-1 system immune checkpoint.
[10] The antibody of [9] above, which is an antibody that binds to PD-L1 or PD-1.
[11] The antibody according to any one of [1] to [10] above, which expresses a targeting molecule.
[12] The antibody of [11] above, wherein the targeting molecule is linked to an uncharged hydrophilic polymer block.
[13] The antibody of [12] above, wherein the targeting molecule is a GLUT1 ligand.
[14] The antibody of [13] above, wherein the GLUT1 ligand is glucose.
[15] The antibody that binds to the immune checkpoint molecule and inhibits the immune checkpoint is an antibody that binds to the immune checkpoint molecule and neutralizes the interaction between the immune checkpoint molecules [1] to The antibody according to any one of [14].
[16] The antibody of [15] above, wherein the immune checkpoint molecule is a counterpart of an immune checkpoint molecule expressed on immune cells.
[17] The antibody of [15] above, wherein the immune checkpoint molecule is an immune checkpoint molecule expressed in immune cells.
[18] A pharmaceutical composition comprising the antibody of any one of [1] to [17] above.
[19] The antibody according to any one of [1] to [6] above, wherein the amino group of the amino acid residue of the antibody is linked to the uncharged hydrophilic polymer block and the environment-responsive bond.
[20] A pharmaceutical composition comprising the antibody of [18] above.
[21] The pharmaceutical composition of [20] above for use in treating cancer.
本発明の抗体の一態様と、当該抗体の生体内における振る舞いを示す。具体的には、上記一態様における抗体は、還元環境応答性のリンカーを介して抗体と非電荷親水性ポリマーブロックとが連結している。抗体は、非電荷親水性ポリマーブロックとの連結により、抗原に対する結合親和性が低減または消失している。これによって、標的臓器以外（非標的組織を含む）においては抗体は不活性化されている。図１では、抗体を脳に送達し、脳において機能させる例が示されている。図１に示されるように、抗体が、例えば、血中から脳に移行すると、脳実質内の還元環境に応答して還元環境応答性のリンカーが開裂する。この開裂は、非電荷親水性ポリマーブロックの抗体からの離脱を生じ、これにより抗体は、非電荷親水性ポリマーブロックによる立体障害から解放され、本来の抗原に対する結合親和性を回復する。このようにして、本発明では、抗体は、脳内および腫瘍組織内、並びにその他のジスルフィド結合が開裂する特別な環境下においてのみ、再活性化し、その抗原に対して作用を発揮し得る。One aspect of the antibody of the present invention and the in vivo behavior of the antibody are shown. Specifically, in the antibody of the above aspect, the antibody and the uncharged hydrophilic polymer block are linked via a reducing environment-responsive linker. Antibodies have reduced or abolished binding affinity for antigens due to linkage with uncharged hydrophilic polymer blocks. As a result, the antibody is inactivated outside the target organ (including non-target tissue). In FIG. 1, an example of antibody delivery to and function in the brain is shown. As shown in FIG. 1, when an antibody, for example, enters the brain from the blood, the reducing-environment-responsive linker is cleaved in response to the reducing environment within the brain parenchyma. This cleavage results in the release of the uncharged hydrophilic polymer block from the antibody, thereby releasing the antibody from steric hindrance by the uncharged hydrophilic polymer block and restoring its original binding affinity for the antigen. Thus, in the present invention, antibodies can be reactivated and exert their action against their antigens only in the brain, in tumor tissue, and other special circumstances where disulfide bonds are cleaved. 本発明の抗体の一態様の具体的模式図を示す。還元環境応答性リンカーを介して、抗ＰＤ－Ｌ１抗体と標的化分子を片末端に有する非電荷親水性ポリマーブロック（例えば、ＰＥＧ）が連結される。1 shows a specific schematic diagram of one embodiment of the antibody of the present invention. An anti-PD-L1 antibody and a non-charged hydrophilic polymer block (eg, PEG) with a targeting molecule at one end are linked via a reduced environment-responsive linker. 修飾された抗体中の未修飾の側鎖アミノ基を定量した結果を示す。修飾後に未修飾の側鎖アミノ基が減少することから非電荷親水性ポリマーブロックは抗体のアミノ基を介して抗体に結合していることが示唆される。2 shows the results of quantification of unmodified side chain amino groups in modified antibodies. The decrease in unmodified side chain amino groups after modification suggests that the uncharged hydrophilic polymer block is attached to the antibody via the amino group of the antibody. 結合親和性を低下させた本発明の修飾抗体が、還元環境下において抗体から修飾部位が除去されてサイズが小さくなり未修飾抗体のサイズにほぼ戻ることを示す（左パネル）。右パネルは、抗体が非電荷親水性ポリマーブロックで修飾されることでゼータ電位を喪失し、還元環境で非電荷親水性ポリマーブロックから解放されることでゼータ電位を回復させることを示す。Modified antibodies of the invention with reduced binding affinity show a reduction in size due to the removal of the modification sites from the antibody in a reducing environment, reverting to approximately the size of the unmodified antibody (left panel). The right panel shows that antibodies are modified with an uncharged hydrophilic polymer block to lose zeta potential and are released from the uncharged hydrophilic polymer block in a reducing environment to restore zeta potential. 本発明の好ましい一態様におけるリンカーの構造と、当該リンカーを用いた場合には、還元環境における当該リンカーの開裂により天然型抗体（未修飾型抗体）が生じる、または放出されることを示す。Figure 2 shows the structure of a linker in a preferred embodiment of the present invention and, when used, cleavage of the linker in a reducing environment yields or releases a native antibody (unmodified antibody). 本発明の抗体の一態様において還元環境依存的にその抗原に対する結合親和性を回復させることを示す。Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１では、抗原に対する結合親和性は実質的に失われている（真ん中のバー参照）が、還元環境下（Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１／ＧＳＨ）では、その結合親和性が未修飾型抗体のレベルにまで回復した。It shows that one embodiment of the antibody of the present invention restores its binding affinity to its antigen in a reducing environment-dependent manner. Gluc-S-aPD-L1 has virtually lost its binding affinity for antigen (see middle bar), but under reducing conditions (Gluc-S-aPD-L1/GSH) its binding affinity recovered to the level of unmodified antibody. 還元環境下で結合性を回復させた抗体が細胞表面のＰＤ－Ｌ１を認識することを示す細胞染色像（上パネル）および結合量（蛍光強度）を示すグラフである。FIG. 4 is a graph showing cell staining images (upper panel) and the amount of binding (fluorescence intensity) showing that an antibody whose binding is restored in a reducing environment recognizes PD-L1 on the cell surface. 抗体の血液中の滞留性（左下パネル）と血漿中の抗体量（右下パネル）を示す。血管内壁に対して未修飾型の抗ＰＤ－Ｌ１抗体が結合を示すのに対して、修飾型抗体は、実質的な結合を示さなかった。Retention of antibody in blood (bottom left panel) and amount of antibody in plasma (bottom right panel) are shown. The unmodified anti-PD-L1 antibody showed binding to the inner wall of blood vessels, whereas the modified antibody showed no substantial binding. 非電荷親水性ポリマーブロックによって修飾された抗体であって、その非電荷親水性ポリマーブロックの０％、２５％、５０％、１００％が片末端にグルコースを有する抗体を得た。図９は、各種抗体の脳腫瘍への蓄積性を示す。Antibodies modified with uncharged hydrophilic polymer blocks were obtained with 0%, 25%, 50%, 100% of the uncharged hydrophilic polymer blocks having glucose at one end. FIG. 9 shows the accumulation of various antibodies in brain tumors. 脳の片側に腫瘍（ＧＬ２６１細胞株）を移植されたマウスにおける腫瘍の増殖に対する各種抗体の抗腫瘍効果を示す。Figure 3 shows the anti-tumor effects of various antibodies on tumor growth in mice implanted with tumors (GL261 cell line) on one side of the brain. 脳の片側に腫瘍（ＧＬ２６１細胞株）を移植されたマウスにおける腫瘍の増殖に対する各種抗体の抗腫瘍効果を示す。左パネルは、生存率を示すカプラン－マイヤー曲線であり、右パネルが処置マウスの体重の変化を示す。Figure 3 shows the anti-tumor effects of various antibodies on tumor growth in mice implanted with tumors (GL261 cell line) on one side of the brain. Left panels are Kaplan-Meier curves showing survival rates and right panels show changes in body weight of treated mice. 脳の片側に腫瘍（ＣＴ－２Ａ細胞株）を移植されたマウスにおける腫瘍の増殖に対する各種抗体の抗腫瘍効果を示す。Antitumor effects of various antibodies on tumor growth in mice implanted with tumors (CT-2A cell line) on one side of the brain. 本発明の一態様の抗体を、二次抗体で検出する系の模式図を示す。図１２では、抗体は、非電荷親水性ポリマーブロックによる修飾によって、二次抗体のアクセスを防ぎ、二次抗体による抗体の認識を許さない。これに対して、還元環境下で、非電荷親水性ポリマーブロックから解放された抗体は、二次抗体により認識される。FIG. 1 shows a schematic diagram of a system for detecting an antibody of one embodiment of the present invention with a secondary antibody. In FIG. 12, the antibody is modified with an uncharged hydrophilic polymer block to prevent access and recognition of the antibody by the secondary antibody. In contrast, in a reducing environment, antibodies released from uncharged hydrophilic polymer blocks are recognized by secondary antibodies. 脳腫瘍内において、本発明の一態様の抗体から修飾が除去されて、本発明の抗体が二次抗体により認識されることを示す。In brain tumors, modifications are removed from the antibody of one aspect of the invention to show that the antibody of the invention is recognized by a secondary antibody. 各種抗体で処置したマウスの脳において、細胞傷害性Ｔ細胞が増加することを示すフローサイトメトリーの結果を示す。Flow cytometry results showing increased cytotoxic T cells in the brains of mice treated with various antibodies. 各種抗体で処置したマウスの脳において、細胞傷害性Ｔ細胞が増加することを示す免疫化学組織学染色の結果を示す。Immunochemical histological staining results showing increased cytotoxic T cells in the brains of mice treated with various antibodies. 各種抗体を投与したマウスにおいて、抗原のプロセッシングおよび提示メカニズムを刺激した後のマウスの血中インターフェロンγ（ＩＦＮγ）の濃度を示す。FIG. 1 shows the concentration of interferon gamma (IFNγ) in blood of mice after stimulation of antigen processing and presentation mechanisms in mice administered with various antibodies. 各種抗体を投与したマウスにおける、免疫抑制性のＦｏｘｐ３＋Ｔ細胞の存在量を示す。The abundance of immunosuppressive Foxp3+ T cells in mice administered with various antibodies is shown. 各種抗体を投与したマウスの脾臓における、ＣＤ４４ｈｉＣＤ６２ＬｌｏｗエフェクターメモリーＴ細胞サブセットの存在量を示す。The abundance of CD44hiCD62Llow effector memory T cell subsets in the spleen of mice treated with various antibodies is shown. 右脳に腫瘍をグラフトされたマウスに対して、本発明の一態様の抗体を投与し、その後、左脳に腫瘍をグラフトとして、その腫瘍を観察するスキームを示す。A scheme is shown in which an antibody of one embodiment of the present invention is administered to a mouse grafted with a tumor in the right brain, then a tumor is grafted in the left brain, and the tumor is observed. 左脳の腫瘍の存在（左パネル）およびカプラン－マイヤー曲線（右パネル）を示す。The presence of tumors in the left brain (left panel) and Kaplan-Meier curves (right panel) are shown. 各種抗体投与後のマウスの非標的組織（肺、腎臓および肝臓）におけるＣＤ４５＋細胞の浸潤を示す。CD45+ cell infiltration in non-target tissues (lung, kidney and liver) of mice after administration of various antibodies is shown. 各種抗体投与後のマウスの非標的組織（肺、腎臓および肝臓）におけるＴＮＦ－α、ＩＬ－６、およびＩＬ－１βレベルを示す。TNF-α, IL-6 and IL-1β levels in non-target tissues (lung, kidney and liver) of mice after administration of various antibodies are shown. 各種抗体投与後のマウスの非標的組織（肺、腎臓および肝臓）において、本発明の一態様の抗体が二次抗体により認識できないこと、すなわち、非標的組織においては抗体から修飾部位が除去されず不活性状態であることを示す。The antibody of one embodiment of the present invention cannot be recognized by a secondary antibody in non-target tissues (lung, kidney, and liver) of mice after administration of various antibodies, that is, the modified site is not removed from the antibody in non-target tissues. Indicates an inactive state. 標的化分子を有しない、非電荷親水性ポリマーブロック（例えば、ＰＥＧ）と還元環境応答性と抗体とがこの順番で連結してなる修飾抗体の模式図を示す。Schematic representation of a modified antibody with no targeting molecule, consisting of an uncharged hydrophilic polymer block (eg, PEG), a reducing environment-responsive polymer, and an antibody linked in that order. がんの皮下移植モデルに対して標的化分子を有しない図２１－１に示される構成を有する修飾抗体を投与したときの抗腫瘍効果を示す。FIG. 21 shows the anti-tumor effect when a modified antibody having the structure shown in FIG. 21-1 without a targeting molecule is administered to a subcutaneous cancer model. 未修飾Ｔ－ＤＭ１とＰＥＧ修飾Ｔ－ＤＭ１の流体力学径の分布を示す。The hydrodynamic diameter distributions of unmodified T-DM1 and PEG-modified T-DM1 are shown. 生体内のがん組織内の還元環境を模擬する還元環境下におけるＰＥＧ修飾抗体の粒径分布の経時的変化と、散乱光強度の変化を示す。Fig. 2 shows temporal changes in the particle size distribution of a PEG-modified antibody under a reducing environment simulating the reducing environment in cancer tissue in vivo and changes in the intensity of scattered light.
発明の具体的な態様Specific aspects of the invention
　本明細書では、「対象」とは、ヒトを含む哺乳動物である。対象は、健常の対象であってもよいし、何らかの疾患に罹患した対象であってもよい。 As used herein, a "subject" is a mammal including a human. A subject may be a healthy subject or a subject suffering from any disease.
　本明細書では、「処置」とは、治療的処置と予防的処置の両方を含む意味で用いられる。本明細書では、「治療」とは、疾患若しくは障害の治療、治癒、防止若しくは、寛解の改善、または、疾患若しくは障害の進行速度の低減を意味する。本明細書では、「予防」とは、疾患もしくは病態の発症の可能性を低下させる、または疾患もしくは病態の発症を遅らせることを意味する。 As used herein, the term "treatment" includes both therapeutic treatment and prophylactic treatment. As used herein, "treatment" means treating, curing, preventing or ameliorating the remission of a disease or disorder, or reducing the rate of progression of a disease or disorder. As used herein, "prevention" means reducing the likelihood of developing a disease or condition or delaying the onset of a disease or condition.
　本明細書では、「疾患」とは、治療が有益な症状を意味する。本明細書では、「がん」とは、一般的には、悪性腫瘍や脳腫瘍などの悪性度の高い腫瘍を意味する。腫瘍は、良性腫瘍および悪性腫瘍を含む。 As used herein, "disease" means a condition for which treatment is beneficial. As used herein, “cancer” generally means highly malignant tumors such as malignant tumors and brain tumors. Tumors include benign and malignant tumors.
　本明細書では、「血液脳関門」とは、血液（または血行）と脳の間に存在して物質の透過に対して選択性を持つ機能的障壁をいう。血液脳関門の実態は、脳血管内皮細胞などであると考えられている。血液脳関門の物質透過性については、不明な点が多いが、グルコース、アルコールおよび酸素は血液脳関門を通過し易いことが知られ、脂溶性物質や小分子（例えば、分子量５００未満）は、水溶性分子や高分子（例えば、分子量５００以上）に比べて通過し易い傾向があると考えられている。多くの脳疾患治療薬や脳診断薬が血液脳関門を通過せず、このことが脳疾患の治療や脳の分析等の大きな障害となっている。本明細書では、「血液神経関門」とは、血行と末梢神経の間に存在して物質の透過に対して選択性を持つ機能的障壁をいう。本明細書では、「血液髄液関門」とは、血行と脳脊髄液との間に存在して物質の透過に対して選択性を持つ機能的障壁をいう。本明細書では、「血液網膜関門」とは、血行と網膜組織の間に存在して物質の透過に対して選択性を持つ機能的障壁をいう。血液神経関門、血液髄液関門および血液網膜関門の実体は、それぞれの関門に存在する血管内皮細胞などであると考えられており、その機能は血液脳関門と同様であると考えられている。 As used herein, the term "blood-brain barrier" refers to a functional barrier that exists between the blood (or blood circulation) and the brain and has selectivity for the permeation of substances. It is believed that the blood-brain barrier is actually composed of cerebrovascular endothelial cells and the like. Although there are many unclear points about the substance permeability of the blood-brain barrier, it is known that glucose, alcohol and oxygen easily pass through the blood-brain barrier, and fat-soluble substances and small molecules (for example, molecular weight less than 500) It is believed that they tend to pass through more easily than water-soluble molecules or macromolecules (eg, molecular weight of 500 or more). Many cerebral disease therapeutic drugs and brain diagnostic drugs do not pass through the blood-brain barrier, and this is a major obstacle to the treatment of cerebral diseases, analysis of the brain, and the like. As used herein, the term "blood-nerve barrier" refers to a functional barrier that exists between the blood circulation and peripheral nerves and has selectivity for the permeation of substances. As used herein, the term "blood-cerebrospinal fluid barrier" refers to a functional barrier that exists between the blood circulation and the cerebrospinal fluid and is selective for the permeation of substances. As used herein, the term "blood-retinal barrier" refers to a functional barrier that exists between the blood circulation and retinal tissue and is selective for the permeation of substances. The blood-nerve barrier, blood-cerebrospinal fluid barrier, and blood-retinal barrier are believed to be vascular endothelial cells and the like present in each barrier, and their functions are believed to be similar to those of the blood-brain barrier.
　本明細書では、「標的化分子」とは、生体内の細胞表面等に存在する標的分子（例えば、細胞表面のタンパク質などの抗原）に対して、生体内のその分子が存在する環境下において結合する分子である。標的化分子は、標的分子に対する結合親和性を有するため、標的化分子を連結した物質を、標的分子の存在位置に能動的に送達（または濃縮する）効果を奏し得る。標的化分子は、細胞表面（例えば、内皮細胞表面）に発現する標的分子に対して結合親和性を有する分子（例えば、抗体、その抗原結合性断片、アプタマー、ペプチド、レクチン等）であり得る。標的化分子を有しない抗体や非電荷親水性ポリマーブロックで修飾した抗体は、例えば、ＥＰＲ効果により受動的に組織（例えば、腫瘍組織）に送達される。標的化分子を有する非電荷親水性ポリマーブロックで修飾した抗体は、生体内の環境下において標的分子に結合できる。本明細書でいう「標的化分子」は、本発明の修飾を受ける抗体部分に連結し得る（好ましくは、非電荷親水性ポリマーブロックの遠位端に結合し得る；ここで近位端とは抗体に連結している側であり、遠位端は、抗体に連結していない側である。）、上記抗体部分とは別の分子部分である。 As used herein, the term "targeting molecule" refers to a target molecule (e.g., an antigen such as a protein on the cell surface) present on the surface of a cell in vivo. It is the molecule that binds. Since the targeting molecule has binding affinity for the target molecule, it can have the effect of actively delivering (or concentrating) the substance linked to the targeting molecule to the location where the target molecule exists. Targeting molecules can be molecules (eg, antibodies, antigen-binding fragments thereof, aptamers, peptides, lectins, etc.) that have binding affinity for target molecules expressed on cell surfaces (eg, endothelial cell surfaces). Antibodies without targeting molecules or modified with uncharged hydrophilic polymer blocks are passively delivered to tissues (eg, tumor tissues) by, for example, the EPR effect. An antibody modified with an uncharged hydrophilic polymer block bearing a targeting molecule can bind to the target molecule in an in vivo environment. As used herein, a "targeting molecule" may be linked to the modified antibody portion of the invention (preferably attached to the distal end of the uncharged hydrophilic polymer block; where the proximal end is the side that is linked to the antibody, and the distal end is the side that is not linked to the antibody), which is a molecular moiety separate from the antibody moiety.
　本明細書では、「ＧＬＵＴ１リガンド」とは、ＧＬＵＴ１と特異的に結合する物質を意
味する。ＧＬＵＴ１リガンドとしては、様々なリガンドが知られ、特に限定されないが例えば、グルコースおよびヘキソースなどの分子が挙げられ、ＧＬＵＴ１リガンドは、いずれも本発明で非電荷親水性ポリマーブロックで修飾した抗体の調製に使用することができる。ＧＬＵＴ１リガンドは、好ましくはＧＬＵＴ１に対してグルコースと同等またはそれ以上の親和性を有する。２－Ｎ－４－（１－アジ－２，２，２－トリフルオロエチル）ベンゾイル－１，３－ビス（Ｄ－マンノース－４－イルオキシ）－２－プロピルアミン（ＡＴＢ－ＢＭＰＡ）、６－（Ｎ－（７－ニトロベンズ－２－オキサ－１，３－ジアゾール－４－イル）アミノ）－２－デオキシグルコース（６－ＮＢＤＧ）、４，６－Ｏ－エチリデン－α－Ｄ－グルコース、２－デオキシ－Ｄ－グルコースおよび３－Ｏ－メチルグルコースもＧＬＵＴ１と結合することが知られ、これらの分子もＧＬＵＴ１リガンドとして本発明に用いることができる。ＧＬＵＴ１リガンドには、ＧＬＵＴ１結合分子が含まれ、ＧＬＵＴ１結合分子としては、ＧＬＵＴ１結合アプタマーが含まれる。ＧＬＵＴ１リガンドは、ＧＬＵＴ１への特異性が高いほど好ましく用いられ得る。ある好ましい例では、ＧＬＵＴ１リガンドは、グルコースであり得る。 As used herein, "GLUT1 ligand" means a substance that specifically binds to GLUT1. Various ligands are known as GLUT1 ligands, including but not limited to molecules such as glucose and hexose, any of which can be used in the preparation of antibodies modified with uncharged hydrophilic polymer blocks in the present invention. can be used. GLUT1 ligands preferably have an affinity for GLUT1 that is equal to or greater than that of glucose. 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-yloxy)-2-propylamine (ATB-BMPA), 6- (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (6-NBDG), 4,6-O-ethylidene-α-D-glucose, 2 -Deoxy-D-glucose and 3-O-methylglucose are also known to bind to GLUT1, and these molecules can also be used as GLUT1 ligands in the present invention. GLUT1 ligands include GLUT1-binding molecules, and GLUT1-binding molecules include GLUT1-binding aptamers. GLUT1 ligands with higher specificity to GLUT1 can be preferably used. In one preferred example, the GLUT1 ligand can be glucose.
　本明細書では、「抗体」は、免疫グロブリンを意味し、一対のジスルフィド結合で安定化された２本の重鎖（Ｈ鎖）と２本の軽鎖（Ｌ鎖）が会合した構造をとるタンパク質をいう。重鎖は、重鎖可変領域ＶＨ、重鎖定常領域ＣＨ１、ＣＨ２、ＣＨ３、及びＣＨ１とＣＨ２の間に位置するヒンジ領域からなり、軽鎖は、軽鎖可変領域ＶＬと軽鎖定常領域ＣＬとからなる。この中で、ＶＨとＶＬからなる可変領域断片（Ｆｖ）が、抗原結合に直接関与し、抗体に多様性を与える領域である。また、ＶＬ、ＣＬ、ＶＨ、ＣＨ１からなる抗原結合領域をＦａｂ領域と呼び、ヒンジ領域、ＣＨ２、ＣＨ３からなる領域をＦｃ領域と呼ぶ。
　可変領域のうち、直接抗原と接触する領域は特に変化が大きく、相補性決定領域（ｃｏｍｐｌｅｍｅｎｔａｒｉｔｙ－ｄｅｔｅｒｍｉｎｉｎｇ　ｒｅｇｉｏｎ：　ＣＤＲ）と呼ばれる。ＣＤＲ以外の比較的変異の少ない部分をフレームワーク（ｆｒａｍｅｗｏｒｋ　ｒｅｇｉｏｎ：　ＦＲ）と呼ぶ。軽鎖と重鎖の可変領域には、それぞれ３つのＣＤＲが存在し、それぞれＮ末端側から順に、重鎖ＣＤＲ１～３及び軽鎖ＣＤＲ１～３と呼ばれる。
　本明細書では、「修飾抗体」とは、化学的修飾を有する抗体を意味する。化学的修飾としては、非電荷親水性ポリマー（例えば、ポリエチレングリコール、ポリオキサゾリン）による修飾などが挙げられる。 As used herein, "antibody" means an immunoglobulin, which has a structure in which two heavy chains (H chains) and two light chains (L chains) stabilized by a pair of disulfide bonds are associated. means protein. The heavy chain consists of a heavy chain variable region VH, heavy chain constant regions CH1, CH2, CH3, and a hinge region located between CH1 and CH2, and the light chain consists of a light chain variable region VL and a light chain constant region CL. consists of Among them, a variable region fragment (Fv) consisting of VH and VL is a region that directly participates in antigen binding and imparts diversity to antibodies. The antigen-binding region consisting of VL, CL, VH and CH1 is called the Fab region, and the region consisting of the hinge region, CH2 and CH3 is called the Fc region.
Among the variable regions, the regions that directly contact the antigen undergo particularly large changes and are called complementarity-determining regions (CDRs). A portion other than the CDRs with relatively few mutations is called a framework region (FR). The light chain and heavy chain variable regions each have three CDRs, which are referred to as heavy chain CDRs 1-3 and light chain CDRs 1-3 in order from the N-terminus.
As used herein, "modified antibody" means an antibody that has a chemical modification. Chemical modifications include modification with uncharged hydrophilic polymers (eg, polyethylene glycol, polyoxazolines).
　本明細書では、抗体は、モノクローナル抗体であっても、ポリクローナル抗体であってもよい。本明細書ではまた、抗体は、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤ、ＩｇＥのいずれのアイソタイプであってもよい。マウス、ラット、ハムスター、モルモット、ウサギ、ニワトリなどの非ヒト動物を免疫して作製したものであってもよいし、組換え抗体であってもよく、キメラ抗体、ヒト化抗体、完全ヒト化抗体等であってもよい。キメラ型抗体とは、異なる種に由来する抗体の断片が連結された抗体をいう。
　「ヒト化抗体」とは、非ヒト由来の抗体に特徴的なアミノ酸配列で、ヒト抗体の対応する位置を置換した抗体を意味し、例えば、マウス又はラットを免疫して作製した抗体の重鎖ＣＤＲ１～３及び軽鎖ＣＤＲ１～３を有し、重鎖及び軽鎖のそれぞれ４つのフレームワーク領域（ＦＲ）を含むその他のすべての領域がヒト抗体に由来するものが挙げられる。かかる抗体は、ＣＤＲ移植抗体と呼ばれる場合もある。用語「ヒト化抗体」は、ヒトキメラ抗体を含む場合もある。
　「ヒトキメラ抗体」は、非ヒト由来の抗体において、非ヒト由来の抗体の定常領域がヒトの抗体の定常領域に置換されている抗体である。
　抗体は、単離されていることができる。抗体は、医薬製品においては、単離されたモノクローナル抗体であることが好ましい。抗体は、抗体依存性細胞傷害活性（ＡＤＣＣ）および／または補体依存性細胞傷害活性（ＣＤＣ）を有し得る。 As used herein, an antibody may be a monoclonal antibody or a polyclonal antibody. Also herein, an antibody can be of any isotype, IgG, IgM, IgA, IgD, IgE. It may be prepared by immunizing non-human animals such as mice, rats, hamsters, guinea pigs, rabbits, chickens, etc., or may be recombinant antibodies, chimeric antibodies, humanized antibodies, fully humanized antibodies. etc. A chimeric antibody refers to an antibody in which antibody fragments derived from different species are linked.
"Humanized antibody" means an antibody in which the corresponding positions of a human antibody are substituted with an amino acid sequence characteristic of a non-human antibody, for example, the heavy chain of an antibody produced by immunizing a mouse or rat Those having CDRs 1-3 and light chain CDRs 1-3, with all other regions derived from human antibodies, including the four framework regions (FRs) each of the heavy and light chains. Such antibodies are sometimes referred to as CDR-grafted antibodies. The term "humanized antibody" may also include human chimeric antibodies.
A “human chimeric antibody” is a non-human antibody in which the constant region of the non-human antibody is replaced with the constant region of a human antibody.
Antibodies can be isolated. The antibody is preferably an isolated monoclonal antibody for pharmaceutical products. Antibodies may have antibody dependent cellular cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC).
　本明細書では、「抗体の抗原結合性断片」とは、抗体のフラグメントであって、抗原への結合性を維持した断片をいう。具体的には、ＶＬ、ＶＨ、ＣＬ及びＣＨ１領域からなるＦａｂおよびヒンジ領域を有するＦａｂ’；２つのＦａｂがヒンジ領域でジスルフィド結合によって連結されているＦ（ａｂ’）２；ＶＬ及びＶＨからなるＦｖ；ＶＬ及びＶＨを人工のポリペプチドリンカーで連結した一本鎖抗体であるｓｃＦｖのほか、ダイアボディ型、ｓｃＤｂ型、タンデムｓｃＦｖ型、ロイシンジッパー型などの多重（または二重）特異性抗体等が挙げられるが、これらに限定されない。 As used herein, the term "antigen-binding fragment of an antibody" refers to a fragment of an antibody that maintains antigen-binding ability. Specifically, Fab' consisting of VL, VH, CL and CH1 regions and Fab' having a hinge region; F(ab')2 in which two Fabs are linked by a disulfide bond at the hinge region; consisting of VL and VH Fv; In addition to scFv, which is a single-chain antibody in which VL and VH are linked by an artificial polypeptide linker, there are multi- (or bi)specific antibodies such as diabodies, scDb, tandem scFv, and leucine zippers. include, but are not limited to.
　本明細書では、「ＣＤＲ」は、抗体の重鎖可変領域および軽鎖可変領域に存在する相補性決定領域である。重鎖および軽鎖可変領域に、それぞれ３つ存在し、Ｎ末端からＣＤＲ１、ＣＤＲ２、およびＣＤＲ３と呼ばれる。ＣＤＲは、例えば、Ｋａｂａｔらの番号付け（Ｋａｂａｔ，Ｅ．Ａ．ｅｔ　ａｌ．，　Ｓｅｑｕｅｎｃｅｓ　ｏｆ　Ｐｒｏｔｅｉｎｓ　ｏｆ　Ｉｍｍｕｎｏｌｏｇｉｃａｌ　Ｉｎｔｅｒｅｓｔ，５ｔｈ　ｅｄ．，　１９９１，　Ｂｅｔｈｅｓｄａ：　ＵＳ　Ｄｅｐｔ．　ｏｆ　Ｈｅａｌｔｈ　ａｎｄ　Ｈｕｍａｎ　Ｓｅｒｖｉｃｅｓ，　ＰＨＳ，　ＮＩＨ．）に基づいて決定され得る。 As used herein, "CDRs" are complementarity determining regions present in the heavy and light chain variable regions of antibodies. There are three each in the heavy and light chain variable regions, designated from the N-terminus as CDR1, CDR2 and CDR3. CDRs are, for example, numbered by Kabat et al. can be determined based on
　本明細書では、「抗原」は、抗体が結合し得る物質をいう。抗原は、免疫原性を有していることができる。抗原は、タンパク質、核酸、および代謝物等であり得る。 As used herein, "antigen" refers to a substance that an antibody can bind to. Antigens can be immunogenic. Antigens can be proteins, nucleic acids, metabolites, and the like.
　本明細書では、「アルキル」とは、直鎖（即ち、分岐でない）若しくは分岐炭素鎖、またはその組み合わせを意味する。アルキルとしては、特に限定されないが例えば、メチル、エチル、ｎ－プロピル、イソプロピル、ｎ－ブチル、ｔ－ブチル、イソブチル、ｎ－ペンチル、ｎ－ヘキシル、ｎ－ヘプチル、およびｎ－オクチル、並びにこれらの異性体が挙げられる。本明細書では、アルキルは、Ｃ1アルキル、Ｃ2アルキル、Ｃ3アルキル、Ｃ4アルキル、Ｃ5アルキル、Ｃ6アルキル、Ｃ7アルキル、またはＣ8アルキルであり得る。これらをまとめて表すときは、Ｃ1－Ｃ8アルキルということがある。
　本明細書では、「アルケニル」とは、アルキルが有する２つの隣接する炭素間に二重結合を有する基を言う。本明細書では、「アルキニル」とは、アルキルが有する２つの隣接する炭素環に三重結合を有する基を言う。
　本明細書では、「低級」とは、炭素数が１～８であることを意味し、例えば、１～６、１～５、１～４、１～３、１～２、２～６、２～５、２～４、または２～３であることを意味し得る。 As used herein, "alkyl" means straight (ie, unbranched) or branched carbon chains, or combinations thereof. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl, and these isomers are included. As used herein, alkyl can be C1 alkyl, C2 alkyl , C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, or C8 alkyl . When collectively representing these, they may be referred to as C 1 -C 8 alkyl.
As used herein, "alkenyl" refers to a group having a double bond between two adjacent carbons of alkyl. As used herein, "alkynyl" refers to the group having triple bonds in two adjacent carbocyclic rings of alkyl.
As used herein, “lower” means having 1 to 8 carbon atoms, such as 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, It can mean 2-5, 2-4, or 2-3.
　本明細書では、「置換されていてもよい」とは、化合物または基が他の基によって置換されていても、置換されていなくてもよい（すなわち、置換または非置換である）ことを意味する。前記他の基としては、ハロゲン、ヒドロキシル基、低級アルキル基、および、好ましくは負の電荷を有する基、例えば、カルボキシル基が挙げられる。 As used herein, "optionally substituted" means that a compound or group may or may not be substituted (i.e., substituted or unsubstituted) by other groups. do. Said other groups include halogens, hydroxyl groups, lower alkyl groups, and preferably negatively charged groups such as carboxyl groups.
　本明細書では、「低血糖状態に制御する」または血糖値を低下させるとは、対象において、その処置がされなければ示したはずの血糖よりも血糖値を低下させることをいう。低血糖は、例えば、倦怠感、手の震え、動悸、頻脈または冷や汗などの自律神経症状が生じない程度の低血糖（例えば、７０ｍｇ／ｄL）以上であり得る。低血糖は、中枢神経症状（例えば、強い脱力感、疲労感、目のかすみ、頭痛、眠気など）および大脳機能低下（例えば、意識レベルの低下、異常行動、痙攣、昏睡など）が生じないレベルであり得る。血糖値は、例えば、８０～１００ｍｇ／ｄＬ程度まで低下させることができる。低血糖を誘発させるとは、空腹時血糖を生じさせることを含む意味で用いられる。低血糖は、例えば、絶食によっても誘発させることができる。低血糖状態に制御する方法としては、糖尿病薬の投与などが挙げられる。例えば、低血糖状態に制御する際に、低血糖状態に制御するという目的を達する限りにおいて、例えば、他の薬剤を摂取し、または水などの飲料を飲むことは許容される。低血糖を誘発させることは、血糖に実質的に影響しない他の処置を
伴ってもよい。 As used herein, "controlling hypoglycemia" or lowering blood glucose levels refers to lowering blood glucose levels in a subject below what they would have had without the treatment. Hypoglycemia can be, for example, a level of hypoglycemia (eg, 70 mg/dL) or higher that does not cause autonomic symptoms such as fatigue, hand tremors, palpitations, tachycardia, or cold sweats. Hypoglycemia is a level that does not cause central nervous system symptoms (e.g., strong weakness, fatigue, blurred vision, headache, drowsiness, etc.) and cerebral dysfunction (e.g., decreased level of consciousness, abnormal behavior, convulsions, coma, etc.). can be The blood sugar level can be lowered, for example, to about 80-100 mg/dL. Inducing hypoglycemia is used in the sense of including producing fasting blood sugar. Hypoglycemia can also be induced, for example, by fasting. Methods for controlling hypoglycemia include administration of antidiabetic drugs. For example, when controlling to a hypoglycemic state, it is permissible, for example, to take other drugs or drink beverages such as water as long as the goal of controlling to a hypoglycemic state is achieved. Inducing hypoglycemia may be accompanied by other treatments that do not substantially affect blood glucose.
　本明細書では、「絶食させる」とは、対象に絶食、例えば、３時間以上、４時間以上、５時間以上、６時間以上、７時間以上、８時間以上、９時間以上、１０時間以上、１１時間以上、１２時間以上、１３時間以上、１４時間以上、１５時間以上、１６時間以上、１７時間以上、１８時間以上、１９時間以上、２０時間以上、２１時間以上、２２時間以上、２３時間以上、２４時間以上、２５時間以上、２６時間以上、２７時間以上、２８時間以上、２９時間以上、３０時間以上、３１時間以上、３２時間以上、３３時間以上、３４時間以上、３５時間以上、３６時間以上、３７時間以上、３８時間以上、３９時間以上、４０時間以上、４１時間以上、４２時間以上、４３時間以上、４４時間以上、４５時間以上、４６時間以上、４７時間以上または４８時間以上の絶食をさせることを意味する。絶食により対象は低血糖を引き起こす。絶食期間は、対象の健康状態に鑑みて医師等により決定され、例えば、対象が空腹時血糖に達する時間以上の期間とすることが好ましい。絶食期間は、例えば、脳血管内皮細胞の血管内表面でのＧＬＵＴ１の発現が増大する、またはプラトーに達する以上の時間としてもよい。絶食期間は、例えば、１２時間以上、２４時間以上または３６時間以上である上記期間とすることができる。また、絶食は、血糖値やＧＬＵＴ１の血管内表面での発現に実質的に影響しない他の処置を伴ってもよい。 As used herein, "fasting" refers to subjecting a subject to fasting, e.g. 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, 19 hours or more, 20 hours or more, 21 hours or more, 22 hours or more, 23 hours 24 hours or more, 25 hours or more, 26 hours or more, 27 hours or more, 28 hours or more, 29 hours or more, 30 hours or more, 31 hours or more, 32 hours or more, 33 hours or more, 34 hours or more, 35 hours or more, 36 hours or more, 37 hours or more, 38 hours or more, 39 hours or more, 40 hours or more, 41 hours or more, 42 hours or more, 43 hours or more, 44 hours or more, 45 hours or more, 46 hours or more, 47 hours or more, or 48 hours It means to fast for more than. Fasting causes the subject to become hypoglycemic. The fasting period is determined by a doctor or the like in view of the subject's health condition, and is preferably set to a period equal to or longer than the time required for the subject to reach fasting blood sugar, for example. The fasting period may be, for example, a period of time during which GLUT1 expression on the intravascular surface of cerebrovascular endothelial cells increases or reaches a plateau. The fasting period can be, for example, the above period of 12 hours or longer, 24 hours or longer, or 36 hours or longer. Fasting may also be accompanied by other treatments that do not substantially affect blood glucose levels or GLUT1 expression on the intravascular surface.
　本明細書では、「血糖値の上昇を誘発させる」とは、低血糖状態に制御した対象、または、低血糖状態を維持させた対象において血糖値を上昇させることをいう。血糖値は、当業者に周知の様々な方法により上昇させることができるが、例えば、血糖値の上昇を誘発するものの投与、例えば、グルコース、フルクトース（果糖）、ガラクトースなどの血糖値の上昇を誘発する単糖の投与、マルトースなどの血糖値の上昇を誘発する多糖の投与、若しくは、デンプンなどの血糖値の上昇を誘発する炭水化物の摂取、または、食事により上昇させることができる。 As used herein, "to induce an increase in blood glucose level" refers to an increase in blood glucose level in a subject controlled to a hypoglycemic state or in a subject maintained in a hypoglycemic state. Blood glucose levels can be raised by various methods well known to those skilled in the art, for example, administration of substances that induce elevation of blood glucose levels, e.g., glucose, fructose (fructose), galactose, etc. It can be increased by administration of monosaccharides that induce blood sugar levels, administration of polysaccharides that induce blood sugar levels such as maltose, intake of carbohydrates that induce blood sugar levels such as starch, or meals.
　本明細書では、「血糖操作」とは、対象に対して、低血糖状態に制御（または維持）し、その後、血糖値を上昇させることをいう。対象に対して低血糖状態に制御した後は、対象の血糖値を低血糖に維持することができる。対象の血糖値を低血糖に維持する時間は、例えば、０時間以上、１時間以上、２時間以上、３時間以上、４時間以上、５時間以上、６時間以上、７時間以上、８時間以上、９時間以上、１０時間以上、１１時間以上、１２時間以上、１３時間以上、１４時間以上、１５時間以上、１６時間以上、１７時間以上、１８時間以上、１９時間以上、２０時間以上、２１時間以上、２２時間以上、２３時間以上、２４時間以上、２５時間以上、２６時間以上、２７時間以上、２８時間以上、２９時間以上、３０時間以上、３１時間以上、３２時間以上、３３時間以上、３４時間以上、３５時間以上、３６時間以上、３７時間以上、３８時間以上、３９時間以上、４０時間以上、４１時間以上、４２時間以上、４３時間以上、４４時間以上、４５時間以上、４６時間以上、４７時間以上、４８時間以上とすることができる。その後、血糖値を上昇させることができる。本明細書では、「血糖を維持する」とは、対象において低血糖を維持するという目的を達する限りにおいて、例えば、他の薬剤を摂取し、または水などの飲料を飲むことは許される。低血糖を誘発させることは、血糖に実質的に影響しない他の処置を伴ってもよい。低血糖は、病的な低血糖ではなく、意識を保つに十分な血糖を有していることが好ましく、例えば、空腹時血糖と同レベルの血糖を意味し得る。低血糖は、後に血糖を上昇させる操作によってＧＬＵＴ１リガンドを表出する修飾抗体がＢＢＢを通過することを促進するために必要なレベルでの低血糖であればよい。 As used herein, "blood sugar manipulation" refers to controlling (or maintaining) a hypoglycemic state in a subject and then increasing the blood sugar level. After controlling the subject to a hypoglycemic state, the subject's blood glucose level can be maintained hypoglycemic. The time to maintain the blood sugar level of the subject at hypoglycemia is, for example, 0 hours or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more , 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, 19 hours or more, 20 hours or more, 21 hours or more, 22 hours or more, 23 hours or more, 24 hours or more, 25 hours or more, 26 hours or more, 27 hours or more, 28 hours or more, 29 hours or more, 30 hours or more, 31 hours or more, 32 hours or more, 33 hours or more , 34 hours or more, 35 hours or more, 36 hours or more, 37 hours or more, 38 hours or more, 39 hours or more, 40 hours or more, 41 hours or more, 42 hours or more, 43 hours or more, 44 hours or more, 45 hours or more, 46 hours or longer, 47 hours or longer, or 48 hours or longer. Blood sugar levels can then be raised. As used herein, "maintaining blood sugar" means, for example, taking other drugs or drinking beverages such as water, as long as the goal of maintaining hypoglycemia in the subject is achieved. Inducing hypoglycemia may be accompanied by other treatments that do not substantially affect blood glucose. Hypoglycemia is preferably not pathologic hypoglycemia, but may mean having sufficient blood sugar to remain conscious, eg, fasting blood sugar levels. Hypoglycemia may be hypoglycemia at a level necessary to facilitate crossing of the BBB by a modified antibody displaying a GLUT1 ligand, which is subsequently manipulated to raise blood sugar.
　ＧＬＵＴ１リガンド（例えば、グルコース）でその外表面を修飾したキャリアは、対象に投与するだけでも脳への蓄積を示す（ＷＯ２０１５／０７５９４２Ａ参照）。従って、本発明による投与計画では、絶食若しくは低血糖を誘発させなくてよく、および／または、血糖値の上昇を誘発させなくてもよい。グルコースが表面に露出するようにグルコースでその外表面を修飾したキャリア、具体的にはミセルまたはポリイオンコンプレックス型ポリマーソーム（ＰＩＣｓｏｍｅ）などの小胞をある投与計画に従って投与すると、顕著にこれらのキャリアが血液脳関門を超えて脳内（脳実質部）に送達される（ＷＯ２０１５／０７５９４２Ａ参照）。従って、本発明による投与計画は、好ましくは、絶食させるか、または低血糖を誘発させた対象に該組成物を投与することを含むが、より好ましくは、本発明による投与計画は、絶食させるか、または低血糖を誘発させた対象に該組成物を投与することおよび該対象において血糖値の上昇を誘発させることを含む。本発明による投与計画では、該組成物は、該対象における血糖値の上昇の誘発と、同時に、連続してまたは逐次的に該対象に投与され得る。投与計画は、該対象への組成物の投与と該対象における血糖値の上昇の誘発との間にインターバルを有してもよいし、有さなくてもよい。該組成物が該対象における血糖値の上昇の誘発と同時に投与される場合には、該組成物は、血糖値の上昇の誘発を引き起こす薬剤と混合した形態で該対象に投与してもよいし、該対象における血糖値の上昇の誘発を引き起こす薬剤とは別の形態で投与してもよい。また、該組成物は、該対象における血糖値の上昇の誘発と、連続してまたは逐次的に該対象に投与される場合には、該組成物は該対象における血糖値の上昇の誘発より前に該対象に投与してもよいし、後に投与してもよいが、好ましくは、該組成物は該対象における血糖値の上昇の誘発より前に該対象に投与することができる。該対象への該組成物の投与よりも先に該対象において血糖値の上昇を誘発させる場合には、該対象において血糖値の上昇を誘発させてから、１時間以内、４５分以内、３０分以内、１５分以内または１０分以内に該対象に該組成物を投与することが好ましい。また、該対象への該組成物の投与よりも後に該対象において血糖値の上昇を誘発させる場合には、該対象に該組成物を投与してから、６時間以内、４時間以内、２時間以内、１時間以内、４５分以内、３０分以内、１５分以内または１０分以内に該対象において血糖値の上昇を誘発させることが好ましい。上記の投与計画のサイクルは、２回以上行なってもよい。グルコース投与とサンプル投与の前後関係は、血液脳関門を通過させるタイミングにより決定することができる。本発明においてグルコースの投与は、食事摂取に変えることができる。ＷＯ２０１５／０７５９４２Ａによれば、血糖操作がなくても、ミセルはＢＢＢを通過して脳内に集積することも明らかになっている。本発明のある態様では、対象は、低血糖状態を誘発されていない。本発明のある態様では、対象は、低血糖状態を誘発されているが、血糖値の上昇の誘発がなされない。 A carrier whose outer surface is modified with a GLUT1 ligand (eg, glucose) shows accumulation in the brain even when administered to a subject (see WO2015/075942A). Thus, dosing regimens according to the invention may not induce fasting or hypoglycemia and/or may not induce elevated blood glucose levels. When a carrier whose outer surface is modified with glucose such that glucose is exposed on the surface, specifically a vesicle such as a micelle or a polyion complex polymersome (PICsome), is administered according to a certain administration regimen, these carriers are remarkably enhanced. It is delivered into the brain (brain parenchyma) across the blood-brain barrier (see WO2015/075942A). Accordingly, dosing regimens according to the present invention preferably comprise administering the composition to a subject who is fasted or hypoglycemic induced, but more preferably dosing regimens according to the present invention include fasting or hypoglycemia-induced subjects. or administering the composition to a subject that has induced hypoglycemia and inducing an increase in blood glucose levels in the subject. In dosing regimens according to the present invention, the composition may be administered to the subject concurrently, sequentially or sequentially with the induction of elevated blood glucose levels in the subject. A dosing regimen may or may not have an interval between administering the composition to the subject and inducing an increase in blood glucose levels in the subject. When the composition is administered at the same time as inducing an increase in blood glucose level in the subject, the composition may be administered to the subject in a form mixed with an agent that induces an increase in blood glucose level. , may be administered in a form separate from the agent that causes the induction of elevated blood glucose levels in the subject. Also, the composition induces an increase in blood glucose levels in the subject and, if administered to the subject sequentially or sequentially, the composition prior to inducing an increase in blood glucose levels in the subject. It may be administered to the subject on or after, but preferably the composition is administered to the subject prior to inducing an increase in blood glucose levels in the subject. within 1 hour, within 45 minutes, or 30 minutes after inducing an increase in blood glucose level in the subject, when an increase in blood glucose level is induced in the subject prior to administration of the composition to the subject Preferably, the composition is administered to the subject within, within 15 minutes, or within 10 minutes. In addition, when an increase in blood glucose level is induced in the subject after administration of the composition to the subject, within 6 hours, within 4 hours, or 2 hours after administration of the composition to the subject Preferably, an increase in blood glucose level is induced in said subject within, within 1 hour, within 45 minutes, within 30 minutes, within 15 minutes or within 10 minutes. Two or more cycles of the above regimen may be performed. The context of glucose administration and sample administration can be determined by the timing of crossing the blood-brain barrier. Glucose administration in the present invention can be replaced with dietary intake. According to WO2015/075942A, it is also revealed that micelles pass through the BBB and accumulate in the brain without glycemic manipulation. In some aspects of the invention, the subject has not been induced to be hypoglycemic. In some aspects of the invention, the subject has been induced to be hypoglycemic, but not induced to have elevated blood glucose levels.
　本発明によれば、修飾されたアミノ基を有する抗体が提供される。
　本発明によれば、修飾は、抗体の抗原に対する結合親和性を低減または消失（喪失）させるものであり得る。このようにすることによって、非標的組織での抗体の機能を低下させ、副作用および有害事象の発生を低減することが期待できる。この結合親和性の低減は、大きいほど好ましく、例えば、修飾された抗体の結合親和性は、未修飾抗体の３０％以下、２５％以下、２０％以下、１５％以下、１０％以下、５％以下、４％以下、３％以下、２％以下、１％以下、または０．１％以下であり得る。結合親和性は、例えば、血清中または生理食塩水中で測定することができる。このように、本発明の抗体は、修飾によって不活性化されていることができる。すなわち、本発明の抗体は、不活性化型抗体であり得る。
　本発明によれば、修飾は、部位特異的な環境応答性であり得る。本発明によればまた、修飾は、還元環境応答性の修飾であり得る。このようにすることで、抗体が脳実質または腫瘍組織の還元環境下において開裂することができ、これによって、抗体の抗原に対する結合親和性を部分的にまたは完全に回復（または再活性化）させることができる。回復、または再活性化は、大きいほど好ましく、例えば、回復または再活性化された抗体の結合親和性は、未修飾抗体の結合親和性の５０％以上、５５％以上、６０％以上、６５％以上、７０％以上、７５％以上、８０％以上、８５％以上、９０％以上、または９５％以上であり得る。このように、本発明の抗体は、修飾は、環境応答性であり、特に還元環境応答性であり、環境依存的に開裂して、抗体の抗原に対する結合親和性を回復または再活性化させることができる。すなわち、本発明の抗体は、環境応答性再活性化可能抗体であり得る。
　本発明によれば、抗体に対する修飾は、特定分子に対する標的化分子による修飾をさらに有していてもよく、当該標的化分子を当該特定分子に対して提示するものであり得る。これによって、抗体を特定分子に標的化することができる。ここで、特定分子は、抗体の抗原とは免疫学的に区別可能に異なる。すなわち、本発明の抗体は、抗原とは免疫学的に区別可能な分子への標的化能を有する、標的化抗体であり得る。
　本発明によれば、例えば、修飾は、抗体に対する修飾は、特定分子に対する標的化分子による修飾をさらに有していてもよく、当該標的化分子を当該特定分子に対して提示するものであり、かつ、修飾は、抗体の抗原に対する結合親和性を低減または消失させる修飾である。特に、本発明の抗体は、修飾によってその抗原に対する結合親和性を低減または消失している。したがって、修飾に、特定抗原に対する標的化能を別に付与し、抗体を標的抗原部位までデリバリーする。本発明によれば、修飾は、部位特異的な環境応答性であり、特に修飾は、還元環境応答性の修飾であり得る。したがって、本発明の抗体は、例えば、抗原とは免疫学的に区別可能な分子への標的化能を有する抗体であって、不活性化型抗体であり、かつ、環境応答性再活性化可能抗体であり得る。
　抗体の抗原に対する結合親和性の低減や消失は、非電荷親水性ポリマーブロックによる立体障害により引き起こされ得る。したがって、非電荷親水性ポリマーブロックを嵩高くすることにより、および／または、修飾率を向上させることによって抗体の抗原に対する結合親和性をより大きく低減させるまたは消失させることができる。 According to the present invention, antibodies with modified amino groups are provided.
According to the present invention, a modification may reduce or abolish (loss) the binding affinity of the antibody for antigen. By doing so, it can be expected that the function of the antibody in non-target tissues will be reduced, and the occurrence of side effects and adverse events will be reduced. This reduction in binding affinity is preferably greater, e.g. or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.1% or less. Binding affinities can be measured, for example, in serum or in saline. Thus, the antibodies of the invention can be inactivated by modification. That is, the antibodies of the present invention can be inactivated antibodies.
According to the invention, the modification may be site-specific and environmentally responsive. Also according to the invention, the modification may be a reducing environment responsive modification. In this way, the antibody can be cleaved in the reducing environment of the brain parenchyma or tumor tissue, thereby partially or fully restoring (or reactivating) the antibody's binding affinity for the antigen. be able to. Restoration or reactivation is preferably greater, e.g., the binding affinity of the restored or reactivated antibody is 50% or more, 55% or more, 60% or more, 65% of that of the unmodified antibody Greater than or equal to 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, or 95% or greater. Thus, the antibodies of the present invention are characterized by modifications that are environmentally responsive, particularly reducing environmentally responsive, that are cleaved in an environmentally dependent manner to restore or reactivate the binding affinity of the antibody for its antigen. can be done. That is, the antibodies of the present invention can be environmentally responsive reactivatable antibodies.
According to the present invention, the modification to the antibody may further comprise modification by a targeting molecule to a specific molecule, and present the targeting molecule to the specific molecule. This allows the antibody to be targeted to a specific molecule. Here, the specific molecule is immunologically distinguishably different from the antigen of the antibody. That is, the antibodies of the present invention can be targeting antibodies capable of targeting molecules that are immunologically distinguishable from antigens.
According to the present invention, for example, a modification to an antibody may further comprise a modification by a targeting molecule to a specific molecule, presenting the targeting molecule to the specific molecule, And the modification is one that reduces or abolishes the binding affinity of the antibody for antigen. In particular, an antibody of the invention has been modified to reduce or eliminate its binding affinity for its antigen. Thus, the modifications are additionally endowed with the ability to target specific antigens and deliver antibodies to the target antigen site. According to the present invention the modification may be a site-specific environmentally responsive modification, in particular the modification may be a reducing environmentally responsive modification. Accordingly, the antibody of the present invention is, for example, an antibody that has the ability to target a molecule that is immunologically distinguishable from an antigen, is an inactivated antibody, and is capable of environmental responsive reactivation. It can be an antibody.
Reduction or loss of binding affinity of antibodies for antigens can be caused by steric hindrance by uncharged hydrophilic polymer blocks. Therefore, the binding affinity of the antibody for the antigen can be reduced or eliminated to a greater extent by increasing the bulk of the uncharged hydrophilic polymer block and/or by increasing the modification rate.
　不活性化型抗体は、抗体のアミノ基の非電荷親水性ポリマーブロック（例えば、ポリエチレングリコールブロックおよびポリオキサゾリンブロック）による修飾により達成され得る。非電荷親水性ポリマーブロックは、抗体の生体適合性や血中滞留性を高め、抗体の標的組織への移行能を向上させ得る。また、非電荷親水性ポリマーブロックは、高い水溶性を有し、抗体の周囲に広がることによって、抗体のその抗原に対するアクセスを抑制することができる。本発明では、抗体の本来の抗原に対する結合親和性を抑制できる非電荷親水性ポリマーブロック（例えば、ポリエチレングリコール）による修飾が好ましく用いられ得る。 Inactivated antibodies can be achieved by modification of antibody amino groups with uncharged hydrophilic polymer blocks (eg, polyethylene glycol blocks and polyoxazoline blocks). The non-charged hydrophilic polymer block can enhance the biocompatibility and blood retention of the antibody, and improve the ability of the antibody to migrate into the target tissue. In addition, the uncharged hydrophilic polymer block has high water solubility and can be spread around the antibody, thereby inhibiting the antibody's access to its antigen. In the present invention, modification with uncharged hydrophilic polymer blocks (eg, polyethylene glycol) that can suppress the binding affinity of antibodies to their original antigens can be preferably used.
　環境応答性再活性化可能抗体は、抗体と上記の非電荷親水性ポリマーブロックとの間に環境応答性に開裂するリンカーを介在させているものであり得る。環境応答性再活性化抗体は、特定の組織の環境に応答して、リンカーを開裂させ、抗体から非電荷親水性ポリマーブロックを切り離し、抗体を遊離させ（または放出させ）、抗体の抗原に対する結合親和性を回復または再活性化させる。環境応答性に開裂するリンカーは、リンカー内に還元環境開裂性の結合（例えば、ジスルフィド結合）を有する。 The environmentally responsive reactivatable antibody may have an environmentally cleavable linker interposed between the antibody and the uncharged hydrophilic polymer block. Environmentally responsive reactivating antibodies respond to the environment of a particular tissue by cleaving the linker, releasing the uncharged hydrophilic polymer block from the antibody, liberating (or releasing) the antibody, and binding the antibody to its antigen. Restores or reactivates affinity. Environmentally cleavable linkers have a reducing environmentally cleavable bond (eg, a disulfide bond) within the linker.
　標的化抗体は、抗体のアミノ基を修飾する非電荷親水性ポリマーブロック（例えば、ポリエチレングリコールブロックおよびポリオキサゾリンブロック）の末端に標的化分子を表出するものであり得る。標的化抗体は、不活性化型抗体を特定組織に標的化する標的化分子を有するものであり得る。 A targeting antibody can display a targeting molecule at the end of an uncharged hydrophilic polymer block (eg, polyethylene glycol block and polyoxazoline block) that modifies the amino groups of the antibody. A targeting antibody can have a targeting molecule that targets the inactivating antibody to a specific tissue.
　本発明によれば、抗体は、例えば、ジスルフィド結合が開裂する特別な環境を標的化した標的化抗体である。ジスルフィド結合が開裂する環境は、例えば、還元環境（例えば、脳実質または腫瘍組織）、または低ｐH条件（例えば、生理学的に可能な低ｐH条件）であり得る。本発明の抗体は、例えば、ジスルフィド結合が開裂する特別な環境下以外では、不活性化型抗体であり、かつ、環境応答性の再活性化可能抗体であり得る。このような抗体を本明細書では、環境標的化型再活性化可能抗体という。例えば、本発明の医薬組成物は、環境標的化型再活性化可能抗体を含む。本発明によれば、抗体は、脳を標的化した標的化抗体であって、不活性化型抗体であり、かつ、還元環境応答性再活性化型抗体であり得る。このような抗体を本明細書では、脳標的化型再活性化可能抗体という。また、本発明によれば、抗体は、腫瘍を標的化した標的化抗体であって、不活性化型抗体であり、かつ、還元環境応答性再活性化型抗体であり得る。このような抗体を本明細書では、腫瘍標的化型再活性化可能抗体という。 According to the present invention, the antibody is, for example, a targeting antibody that targets a special environment in which disulfide bonds are cleaved. The environment in which disulfide bonds are cleaved can be, for example, a reducing environment (eg, brain parenchyma or tumor tissue), or low pH conditions (eg, physiologically possible low pH conditions). The antibody of the present invention can be an inactivated antibody and an environmentally responsive reactivatable antibody, for example, except under special circumstances in which disulfide bonds are cleaved. Such antibodies are referred to herein as environmental targeting reactivatable antibodies. For example, the pharmaceutical compositions of the invention include environmental targeting reactivatable antibodies. According to the present invention, the antibody can be a brain-targeted targeting antibody, an inactivating antibody, and a reactivating antibody responsive to a reducing environment. Such antibodies are referred to herein as brain-targeted reactivatable antibodies. Further, according to the present invention, the antibody can be a tumor-targeting antibody, an inactivating antibody, and a reactivating antibody responsive to a reducing environment. Such antibodies are referred to herein as tumor-targeting reactivatable antibodies.
　本発明によれば、例えば、標的化分子でその末端が修飾されたポリエチレングリコールブロックが、環境応答性の結合（例えば、還元環境応答性の結合、例えば、ジスルフィド結合）を介して、抗体のアミノ基を修飾している。すなわち、抗体は、標的化分子でその末端が修飾された非電荷親水性ポリマーブロック（例えば、ポリエチレングリコールブロック）が、環境応答性の結合（例えば、還元環境応答性の結合、例えば、ジスルフィド結合）を介して連結したリンカーで修飾されたアミノ基を有し得る。本発明によれば、このような抗体が提供され得る。 According to the present invention, for example, a polyethylene glycol block modified at its ends with a targeting molecule is attached to an amino acid of an antibody via an eco-responsive bond (e.g., a reducing eco-responsive bond, e.g., a disulfide bond). modifying the group. That is, an antibody consists of an uncharged hydrophilic polymer block (e.g., a polyethylene glycol block) modified at its ends with a targeting molecule to form an environmentally responsive bond (e.g., a reduced environmentally responsive bond, e.g., a disulfide bond). can have amino groups modified with linkers connected via According to the invention, such antibodies may be provided.
　本発明によれば、抗体は、がん抗原に対して結合する抗体であり得る。本発明によれば、抗体は、免疫チェックポイント分子のいずれかに結合する抗体であり得る。ある態様では、抗体は、ＬＬ１（抗ＣＤ７４抗体）、ＬＬ２またはＲＦＢ４（抗ＣＤ２２抗体）、ベルツズマブ（ｈＡ２０、抗ＣＤ２０抗体）、リツキシマブ（ｒｉｔｕｘｕｍａｂ）（抗ＣＤ２０抗体）、オビヌツズマブ（ＧＡ１０１、抗ＣＤ２０抗体）、ランブロリズマブ（抗ＰＤ－１ 受容体抗体）、ニボルマブ（抗ＰＤ－１ 受容体抗体）、イピリムマブ（抗ＣＴＬＡ－４抗体）、ＲＳ７（抗上皮糖タンパク質－１（ＥＧＰ－１、ＴＲＯＰ－２としても知られる）抗体）、ＰＡＭ４またはＫＣ４（両方とも抗ムチン）、ＭＮ－１４（抗癌胎児性抗原（ＣＥＡ、ＣＤ６６ｅもしくはＣＥＡＣＡＭ５としても知られる）、ＭＮ－１５またはＭＮ－３（抗ＣＥＡＣＡＭ６抗体）、Ｍｕ－９（抗結腸特異抗原－ｐ抗体）、Ｉｍｍｕ ３１（抗α－フェトプロテイン抗体）、Ｒ１（抗ＩＧＦ－１Ｒ抗体）、Ａ１９（抗ＣＤ１９抗体）、ＴＡＧ－７２（例えばＣＣ４９抗体）、Ｔｎ、Ｊ５９１またはＨｕＪ５９１（抗ＰＳＭＡ（前立腺特異的膜抗原）抗体、ＡＢ－ＰＧ１－ＸＧ１－０２６（抗ＰＳＭＡ 二量体抗体）、Ｄ２／Ｂ（抗ＰＳＭＡ抗体）、Ｇ２５０（抗炭酸脱水酵素ＩＸ ＭＡｂ抗体）、Ｌ２４３（抗ＨＬＡ－ＤＲ抗体）アレムツズマブ（抗ＣＤ５２抗体）、ベバシズマブ（抗ＶＥＧＦ抗体）、セツキシマブ（抗ＥＧＦＲ抗体）、ゲムツズマブ（抗ＣＤ３３抗体）、イブリツモマブチウキセタン（抗ＣＤ２０抗体）；パニツムマブ（抗ＥＧＦＲ抗体）；トシツモマブ（抗ＣＤ２０抗体）；ＰＡＭ４（アカテトラキセタン（ａｋａ ｃｌｉｖａｔｕｚｕｍａｂ）、抗ムチン抗体）およびトラスツズマブ（抗ＥｒｂＢ２抗体）が挙げられる。このような抗体は当業者に公知である（例えば、米国特許第５，６８６，０７２号；第５，８７４，５４０号；第６，１０７，０９０号；第６，１８３，７４４号；第６，３０６，３９３号；第６，６５３，１０４号；第６，７３０．３００号；第６，８９９，８６４号；第６，９２６，８９３号；第６，９６２，７０２号；第７，０７４，４０３号；第７，２３０，０８４号；第７，２３８，７８５号；第７，２３８，７８６号；第７，２５６，００４号；第７，２８２，５６７号；第７，３００，６５５号；第７，３１２，３１８号；第７，５８５，４９１号；第７，６１２，１８０号；第７，６４２，２３９号および米国特許出願公開公報第２００５０２７１６７１号；第２００６０１９３８６５号；第２００６０２１０４７５号；第２００７００８７００１号を参照。これらの各実施例部分は本明細書に援用される）。 According to the present invention, the antibody can be an antibody that binds to a cancer antigen. According to the invention, the antibody can be an antibody that binds to any of the immune checkpoint molecules. In certain aspects, the antibody is LL1 (anti-CD74 antibody), LL2 or RFB4 (anti-CD22 antibody), veltuzumab (hA20, anti-CD20 antibody), rituxumab (anti-CD20 antibody), obinutuzumab (GA101, anti-CD20 antibody) , lambrolizumab (anti-PD-1 receptor antibody), nivolumab (anti-PD-1 receptor antibody), ipilimumab (anti-CTLA-4 antibody), RS7 (anti-epithelial glycoprotein-1 (EGP-1, also known as TROP-2 ), PAM4 or KC4 (both anti-mucin), MN-14 (anti-carcinoembryonic antigen (also known as CEA, CD66e or CEACAM5), MN-15 or MN-3 (anti-CEACAM6 antibody), Mu-9 (anti-colon specific antigen-p antibody), Immu 31 (anti-α-fetoprotein antibody), R1 (anti-IGF-1R antibody), A19 (anti-CD19 antibody), TAG-72 (e.g. CC49 antibody), Tn, J591 or HuJ591 (anti-PSMA (prostate-specific membrane antigen) antibody, AB-PG1-XG1-026 (anti-PSMA dimer antibody), D2/B (anti-PSMA antibody), G250 (anti-carbonic anhydrase IX MAb antibody) , L243 (anti-HLA-DR antibody) alemtuzumab (anti-CD52 antibody), bevacizumab (anti-VEGF antibody), cetuximab (anti-EGFR antibody), gemtuzumab (anti-CD33 antibody), ibritumomab tiuxetan (anti-CD20 antibody); panitumumab ( tositumomab (anti-CD20 antibody), PAM4 (aka clivatuzumab, anti-mucin antibody) and trastuzumab (anti-ErbB2 antibody) Such antibodies are known to those skilled in the art ( For example, U.S. Patent Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,730,300; 6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,612,180; 7,642,239 and U.S. Patent Application Publication No. 20050271671; 060193865; 20060210475; 20070087001. The Examples section of each of these is hereby incorporated by reference).
　また、使用される特定の既知の抗体は、ｈＰＡＭ４（米国特許第７，２８２，５６７号）、ｈＡ２０（米国特許第７，２５１，１６４号）、ｈＡ１９（米国特許第７，１０９，３０４号）、ｈＩＭＭＵ－３１（米国特許第７，３００，６５５号）、ｈＬＬ１（米国特許第７，３１２，３１８号）、ｈＬＬ２（米国特許第７，０７４，４０３号）、ｈＭｕ－９（米国特許第７，３８７，７７３号）、ｈＬ２４３（米国特許第７，６１２，１８０号）、ｈＭＮ－１４（米国特許第６，６７６，９２４号）、ｈＭＮ－１５（米国特許第７，５４１，４４０号）、ｈＲ１（米国特許出願公開公報第１２／７７２，６４５号）、ｈＲＳ７（米国特許第７，２３８，７８５号）、ｈＭＮ－３（米国特許第７，５４１，４４０号）、ＡＢ－ＰＧ１－ＸＧ１－０２６（米国特許出願公開公報第１１／９８３，３７２号、ＡＴＣＣ ＰＴＡ－４４０５およびＰＴＡ－４４０６として表される）、およびＤ２／Ｂ（国際特許公開公報第２００９／１３０５７５号）を含み、これらの引用した特許または公開公報の全体は参照により本明細書に援用される。また、限定するものではないが、腫瘍関連抗原を含む多様な疾患標的に対する多くの抗体は、ＡＴＣＣに蓄積されており、かつ／または公開されている可変領域配列を有し、請求される方法および組成物において使用するために入手可能である。例えば、米国特許第７，３１２，３１８号；第７，２８２，５６７号；第７，１５１，１６４号；第７，０７４，４０３号；第７，０６０，８０２号；第７，０５６，５０９号；第７，０４９，０６０号；第７，０４５，１３２号；第７，０４１，８０３号；第７，０４１，８０２号；第７，０４１，２９３号；第７，０３８，０１８号；第７，０３７，４９８号；第７，０１２，１３３号；第７，００１，５９８号；第６，９９８，４６８号；第６，９９４，９７６号；第６，９９４，８５２号；第６，９８９，２４１号；第６，９７４，８６３号；第６，９６５，０１８号；第６，９６４，８５４号；第６，９６２，９８１号；第６，９６２，８１３号；第６，９５６，１０７号；第６，９５１，９２４号；第６，９４９，２４４号；第６，９４６，１２９号；第６，９４３，０２０号；第６，９３９，５４７号；第６，９２１，６４５号；第６，９２１，６４５号；第６，９２１，５３３号；第６，９１９，４３３号；第６，９１９，０７８号；第６，９１６，４７５号；第６，９０５，６８１号；第６，８９９，８７９号；第６，８９３，６２５号；第６，８８７，４６８号；第６，８８７，４６６号；第６，８８４，５９４号；第６，８８１，４０５号；第６，８７８，８１２号；第６，８７５，５８０号；第６，８７２，５６８号；第６，８６７，００６号；第６，８６４，０６２号；第６，８６１，５１１号；第６，８６１，２２７号；第６，８６１，２２６号；第６，８３８，２８２号；第６，８３５，５４９号；第６，８３５，３７０号；第６，８２４，７８０号；第６，８２４，７７８号；第６，８１２，２０６号；第６，７９３，９２４号；第６，７８３，７５８号；第６，７７０，４５０号；第６，７６７，７１１号；第６，７６４，６８８号；第６，７６４，６８１号；第６，７６４，６７９号；第６，７４３，８９８号；第６，７３３，９８１号；第６，７３０，３０７号；第６，７２０，１５５号；第６，７１６，９６６号；第６，７０９，６５３号；第６，６９３，１７６号；第６，６９２，９０８号；第６，６８９，６０７号；第６，６８９，３６２号；第６，６８９，３５５号；第６，６８２，７３７号；第６，６８２，７３６号；第６，６８２，７３４号；第６，６７３，３４４号；第６，６５３，１０４号；第６，６５２，８５２号；第６，６３５，４８２号；第６，６３０，１４４号；第６，６１０，８３３号；第６，６１０，２９４号；第６，６０５，４４１号；第６，６０５，２７９号；第６，５９６，８５２号；第６，５９２，８６８号；第６，５７６，７４５号；第６，５７２；８５６号；第６，５６６，０７６号；第６，５６２，６１８号；第６，５４５，１３０号；第６，５４４，７４９号；第６，５３４，０５８号；第６，５２８，６２５号；第６，５２８，２６９号；第６，５２１，２２７号；第６，５１８，４０４号；第６，５１１，６６５号；第６，４９１，９１５号；第６，４８８，９３０号；第６，４８２，５９８号；第６，４８２，４０８号；第６，４７９，２４７号；第６，４６８，５３１号；第６，４６８，５２９号；第６，４６５，１７３号；第６，４６１，８２３号；第６，４５８，３５６号；第６，４５５，０４４号；第６，４５５，０４０号，第６，４５１，３１０号；第６，４４４，２０６号；第６，４４１，１４３号；第６，４３２，４０４号；第６，４３２，４０２号；第６，４１９，９２８号；第６，４１３，７２６号；第６，４０６，６９４号；第６，４０３，７７０号；第６，４０３，０９１号；第６，３９５，２７６号；第６，３９５，２７４号；第６，３８７，３５０号；第６，３８３，７５９号；第６，３８３，４８４号；第６，３７６，６５４号；第６，３７２，２１５号；第６，３５９，１２６号；第６，３５５，４８１号；第６，３５５，４４４号；第６，３５５，２４５号；第６，３５５，２４４号；第６，３４６，２４６号；第６，３４４，１９８号；第６，３４０，５７１号；第６，３４０，４５９号；第６，３３１，１７５号；第６，３０６，３９３号；第６，２５４，８６８号；第６，１８７，２８７号；第６，１８３，７４４号；第６，１２９，９１４号；第６，１２０，７６７号；第６，０９６，２８９号；第６，０７７，４９９号；第５，９２２，３０２号；第５，８７４，５４０号；第５，８１４，４４０号；第５，７９８，２２９号；第５，７８９，５５４号；第５，７７６，４５６号；第５，７３６，１１９号；第５，７１６，５９５号；第５，６７７，１３６号；第５，５８７，４５９号；第５，４４３，９５３号；第５，５２５，３３８号を参照されたい。これら文献の全体は参照により本明細書に援用される。これらは単なる例示であり、多様な他の抗体およびそれらのハイブリドーマが当業者に公知である。ほとんどの疾患関連抗原のいずれかに対する抗体の配列または抗体－分泌ハイブリドーマは、対象である選択した疾患関連標的に対する抗体を、ＡＴＣＣ、ＮＣＢＩ、および／またはＵＳＰＴＯで単純に検索することにより入手し得ることを当業者は理解している。クローン化した抗体の抗原結合ドメインは、当業者に公知である標準的な技術を使用して増幅し、切断し、発現ベクターに連結し、適合した宿主細胞に遺伝子導入されてもよく、かつ、タンパク質産生のために使用されてもよい（例えば、米国特許第７，５３１，３２７号；第７，５３７，９３０号；第７，６０８，４２５号および第７，７８５，８８０号を参照。これら文献の全体は参照により本明細書に援用される）。 Also, specific known antibodies of use are hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164), hA19 (U.S. Pat. No. 7,109,304) , hIMMU-31 (US Pat. No. 7,300,655), hLL1 (US Pat. No. 7,312,318), hLL2 (US Pat. No. 7,074,403), hMu-9 (US Pat. No. 7 , 387,773), hL243 (US Pat. No. 7,612,180), hMN-14 (US Pat. No. 6,676,924), hMN-15 (US Pat. No. 7,541,440), hR1 (U.S. Patent Application Publication No. 12/772,645), hRS7 (U.S. Patent No. 7,238,785), hMN-3 (U.S. Patent No. 7,541,440), AB-PG1-XG1- 026 (denoted as US Patent Application Publication No. 11/983,372, ATCC PTA-4405 and PTA-4406), and D2/B (International Patent Publication No. 2009/130575), citing these The entirety of the patents or publications issued to this patent are hereby incorporated by reference. Also, many antibodies directed against a variety of disease targets, including but not limited to tumor-associated antigens, have been deposited in the ATCC and/or have variable region sequences published and are subject to the claimed methods and Available for use in compositions. 7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,875,580; 6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,226; 6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,812,206; 6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,681; 6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; No. 6,635,482; 6,630,1 44; 6,610,833; 6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,576,745; 6,572; 856; 6,566,076; 6,562,618; 6,545,130; 6,534,058; 6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468, 6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310 6,444,206; 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481; 6,355,244; 6,346,246; 6,344,198; 6,340,571; 6,340,459 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540; 798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595; 459; 5,443,953; 5,525,338. These documents are incorporated herein by reference in their entirety. These are merely examples and a variety of other antibodies and hybridomas thereof are known to those skilled in the art. The sequences of antibodies or antibody-secreting hybridomas to any of most disease-associated antigens can be obtained by simply searching the ATCC, NCBI, and/or USPTO for antibodies to the selected disease-associated target of interest. are understood by those skilled in the art. The antigen-binding domain of the cloned antibody may be amplified using standard techniques known to those of skill in the art, cleaved, ligated into an expression vector, transfected into a suitable host cell, and may be used for protein production (see, e.g., U.S. Patent Nos. 7,531,327; 7,537,930; 7,608,425 and 7,785,880; these the entirety of which is incorporated herein by reference).
　本発明によれば、標的分子（例えば、抗原）としては、例えば、炭酸脱水酵素ＩＸ、Ｂ７、ＣＣＣＬ１９、ＣＣＣＬ２１、ＣＳＡｐ、ＨＥＲ－２／ｎｅｕ、ＢｒＥ３、ＣＤ１、ＣＤ１ａ、ＣＤ２、ＣＤ３、ＣＤ４、ＣＤ５、ＣＤ８、ＣＤ１１Ａ、ＣＤ１４、ＣＤ１５、ＣＤ１６、ＣＤ１８、ＣＤ１９、ＣＤ２０（例えば、Ｃ２Ｂ８、ｈＡ２０、１Ｆ５　ＭＡｂｓ）、ＣＤ２１、ＣＤ２２、ＣＤ２３、ＣＤ２５、ＣＤ２９、ＣＤ３０、ＣＤ３２ｂ、ＣＤ３３、ＣＤ３７、ＣＤ３８、ＣＤ４０、ＣＤ４０Ｌ、ＣＤ４４、ＣＤ４５、ＣＤ４６、ＣＤ４７、ＣＤ５２、ＣＤ５４、ＣＤ５５、ＣＤ５９、ＣＤ６４、ＣＤ６７、ＣＤ７０、ＣＤ７４、ＣＤ７９ａ、ＣＤ８０、ＣＤ８３、ＣＤ９５、ＣＤ１２６、ＣＤ１３３、ＣＤ１３８、ＣＤ１４７、ＣＤ１５４、ＣＥＡＣＡＭ５、ＣＥＡＣＡＭ６、ＣＴＬＡ－４、α－フェトプロテイン（ＡＦＰ）、ＶＥＧＦ、フィブロネクチンスプライシング変異体、ＥＤ－Ｂ　フィブロネクチン（例えば、Ｌ１９）、ＥＧＰ－１（ＴＲＯＰ－２）、ＥＧＰ－２（例えば、１７－１Ａ）、ＥＧＦ受容体（ＥｒｂＢ１）、ＥｒｂＢ２、ＥｒｂＢ３、Ｈ因子、ＦＨＬ－１、Ｆｌｔ－３、葉酸受容体、Ｇａ７３３、ＧＲＯ－β、ＨＭＧＢ－１、低酸素誘導因子（ＨＩＦ）、ＨＭ１．２４、ＨＥＲ－２／ｎｅｕ、ヒストンＨ２Ｂ、ヒストンＨ３、ヒストンＨ４、インスリン様増殖因子（ＩＬＧＦ）、ＩＦＮ－γ、ＩＦＮ－α、ＩＦＮ－β、ＩＦＮ－λ、ＩＬ－２Ｒ、ＩＬ－４Ｒ、ＩＬ－６Ｒ、ＩＬ－１３Ｒ、ＩＬ－１５Ｒ、ＩＬ－１７Ｒ、ＩＬ－１８Ｒ、ＩＬ－２、ＩＬ－６、ＩＬ－８、ＩＬ－１２、ＩＬ－１５、ＩＬ－１７、ＩＬ－１８、ＩＬ－２５、ＩＰ－１０、ＩＧＦ－１Ｒ、Ｉａ、ＨＭ１．２４、ガングリオシド、ＨＣＧ、Ｌ２４３に結合するＨＬＡ－ＤＲ抗原、ＣＤ６６抗原、すなわちＣＤ６６ａ－ｄまたはそれらの組み合わせ、ＭＡＧＥ、ｍＣＲＰ、ＭＣＰ－１、ＭＩＰ－１Ａ、ＭＩＰ－１Ｂ、マクロファージ遊走阻止因子（ＭＩＦ）、ＭＵＣ１、ＭＵＣ２、ＭＵＣ３、ＭＵＣ４、ＭＵＣ５ａｃ、胎盤増殖因子（ＰｌＧＦ）、ＰＳＡ（前立腺特異抗原）、ＰＳＭＡ、ＰＡＭ４抗原、ＰＤ－１受容体、ＰＤ－Ｌ１、ＮＣＡ－９５、ＮＣＡ－９０、Ａ３、Ａ３３、Ｅｐ－ＣＡＭ、ＫＳ－１、Ｌｅ（ｙ）、メソセリン、Ｓ１００、テネイシン、ＴＡＣ、Ｔｎ抗原、Ｔｈｏｍａｓ－Ｆｒｉｅｄｅｎｒｅｉｃｈ抗原、腫瘍壊死抗原、腫瘍血管新生抗原、ＴＮＦ－α、ＴＲＡＩＬ受容体（Ｒ１およびＲ２）、ＴＲＯＰ－２、ＶＥＧＦＲ、ＲＡＮＴＥＳ、Ｔ１０１、ならびに癌幹細胞抗原、補体因子、Ｃ３、Ｃ３ａ、Ｃ３ｂ、Ｃ５ａ、Ｃ５、ならびに癌遺伝子産物が挙げられる。また、標的化分子は、これらに結合する抗体、その抗原結合性断片、リガンド、アプタマー、またはペプチドであり得る。 According to the present invention, target molecules (eg, antigens) include, for example, carbonic anhydrase IX, B7, CCCL19, CCCL21, CSAp, HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5 , CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L , CD44, CD45, CD46, CD47, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM6, CTLA-4 , α-fetoprotein (AFP), VEGF, fibronectin splice variants, ED-B fibronectin (eg, L19), EGP-1 (TROP-2), EGP-2 (eg, 17-1A), EGF receptor (ErbB1 ), ErbB2, ErbB3, Factor H, FHL-1, Flt-3, folate receptor, Ga733, GRO-β, HMGB-1, hypoxia-inducible factor (HIF), HM1.24, HER-2/neu, histones H2B, histone H3, histone H4, insulin-like growth factor (ILGF), IFN-γ, IFN-α, IFN-β, IFN-λ, IL-2R, IL-4R, IL-6R, IL-13R, IL- 15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, IGF-1R, Ia, HM1.24, gangliosides, HCG, HLA-DR antigen binding to L243, CD66 antigen ie CD66a-d or combinations thereof, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophage migration inhibition factor (MIF), MUC1, MUC2, MUC3, MUC4, MUC5ac, placental growth factor (PlGF), PSA (prostate specific antigen), PSMA, PAM4 antigen, PD-1 receptor, PD-L1, NCA-95, NCA- 90, A3, A33, Ep-CAM, KS-1, Le(y), mesothelin, S100, tenascin, TAC, Tn antigen, Thomas-Friedenrei ch antigen, tumor necrosis antigen, tumor angiogenesis antigen, TNF-α, TRAIL receptors (R1 and R2), TROP-2, VEGFR, RANTES, T101, and cancer stem cell antigens, complement factors, C3, C3a, C3b, C5a, C5, as well as oncogene products. Targeting molecules can also be antibodies, antigen-binding fragments thereof, ligands, aptamers, or peptides that bind to them.
　ある態様では、抗体は、抗原に特異的に結合することができる。ある態様では、抗体は、抗原に対して、１０－８Ｍ、１０－９Ｍ、１０－１０Ｍ、１０－１１Ｍ、または１０－１２Ｍ以下の結合親和性（ＫＤ＝ｋｏｆｆ／ｋｏｎ）を有し得る。ある態様では、抗体は、抗原に対して、１０－１５Ｍ以上、Ｍ以上、１０－１４Ｍ以上、１０－１３Ｍ以上、１０－１２Ｍ以上、１０－１１Ｍ以上、または１０－１０Ｍ以上の結合親和性を有し得る。ある態様では、抗体は、抗原に対して、１０－８Ｍ～１０－１５Ｍ、１０－９Ｍ～１０－１４Ｍ、１０－１０Ｍ～１０－１３Ｍ、もしくは１０－１１Ｍ～１０－１２Ｍ、または上記上限値のいずれかと下限値のいずれかの間の結合親和性を有し得る。ある態様では、修飾抗体は、抗原に対して、１０－６Ｍ以上、１０－５Ｍ以上、１０－４Ｍ以上、１０－３Ｍ以上、１０－２Ｍ以上、または検出限界以下の結合親和性を有し得る。ある態様では、修飾抗体は、抗原に対して、１０－６Ｍ～１０－１Ｍ、１０－４Ｍ～１０－１Ｍ、１０－３Ｍ～１０－１Ｍ、または１０－２Ｍ～１０－１Ｍの間の、または検出限界以下の結合親和性を有し得る。ある態様では、抗体は、抗原に対して、上記の抗体の結合親和性を有し、かつ、修飾抗体は、抗原に対して、上記の修飾抗体の結合親和性を有し得る。 In some aspects, the antibody can specifically bind to an antigen. In certain aspects, the antibody has a binding affinity for the antigen of 10 −8 M, 10 −9 M, 10 −10 M, 10 −11 M, or 10 −12 M or less (KD=k off /k on ). In some embodiments, the antibody is 10 −15 M or greater, 10 −14 M or greater, 10 −13 M or greater, 10 −12 M or greater, 10 −11 M or greater, or 10 −10 M or greater to the antigen. or higher binding affinity. In some embodiments, the antibody is 10 −8 M to 10 −15 M, 10 −9 M to 10 −14 M, 10 −10 M to 10 −13 M, or 10 −11 M to 10 −1 M to the antigen. It may have a binding affinity of 12 M, or between any of the above upper and lower limits. In some embodiments, the modified antibody has a binding affinity for the antigen of 10 −6 M or greater, 10 −5 M or greater, 10 −4 M or greater, 10 −3 M or greater, 10 −2 M or greater, or an undetectable binding affinity. can have gender. In certain aspects, the modified antibody has a concentration of 10 −6 M to 10 −1 M, 10 −4 M to 10 −1 M, 10 −3 M to 10 −1 M, or 10 −2 M to 10 −1 M to the antigen. It may have a binding affinity between -1 M or below the limit of detection. In certain aspects, the antibody has the binding affinity for the antigen of the antibody described above, and the modified antibody can have the binding affinity for the antigen of the modified antibody described above.
　本発明によれば、標的化分子はＧＬＵＴ１リガンドであり得る。すなわち、本発明によれば、ＧＬＵＴ１リガンドにより修飾された抗体であって、ＧＬＵＴ１リガンド、非電荷親水性ポリマーブロック、環境応答性結合、および抗体が、この順番で連結した抗体が提供される。環境応答性結合は、脳実質または腫瘍組織の還元環境下で開裂する結合である。環境応答性の結合は、血中では安定であり得る。したがって、本発明の上記抗体は、血中に投与された場合であっても、ＧＬＵＴ１により修飾された抗体であって、ＧＬＵＴ１リガンド、非電荷親水性ポリマーブロック、環境応答性結合、および抗体が、この順番で連結した抗体の存在形態を維持している。そして、脳実質または腫瘍組織に取り込まれると、環境応答性結合が開裂して、非電荷親水性ポリマーブロックが抗体から分離される。本発明では、修飾は抗体のアミノ基に対して行われ得る。 According to the invention, the targeting molecule can be a GLUT1 ligand. That is, the present invention provides an antibody modified with a GLUT1 ligand, wherein the GLUT1 ligand, an uncharged hydrophilic polymer block, an environment-responsive bond, and an antibody are linked in that order. Environmentally responsive bonds are bonds that cleave under the reducing environment of brain parenchyma or tumor tissue. Environmentally responsive binding can be stable in blood. Thus, the antibody of the present invention is a GLUT1-modified antibody, even when administered into the blood, wherein the GLUT1 ligand, the uncharged hydrophilic polymer block, the environment-responsive bond, and the antibody are This order maintains the form of existence of the ligated antibody. Then, upon internalization into the brain parenchyma or tumor tissue, the environmentally responsive bonds are cleaved to separate the uncharged hydrophilic polymer block from the antibody. In the present invention modifications may be made to the amino groups of the antibody.
　本発明によれば、
　ＧＬＵＴ１リガンド、非電荷親水性ポリマーブロック、環境応答性結合、および抗体がこの順番で連結された複合体形態の抗体が提供される。本発明によれば、非電荷親水性ポリマーブロック－環境応答性結合－抗体の構造を有する修飾抗体が提供される。ここで、記号「－」は、結合またはスペーサーを表し、当該記号の前に記載された要素と後に記載された要素とが結合またはスペーサーを介して連結していることを意味する。スペーサーは、生体内において安定な化学的性質を有するものである。本発明のある態様では、環境応答性結合は、還元環境応答性結合であり、例えば、ジスルフィド結合である。本発明では、ＧＬＵＴ１リガンドは、グルコースであり得る。グルコースは、ＧＬＵＴ１と結合可能に、非電荷親水性ポリマーブロックと連結している。 According to the invention,
An antibody in the form of a conjugate is provided in which a GLUT1 ligand, an uncharged hydrophilic polymer block, an environment-responsive bond, and an antibody are linked in that order. According to the present invention, a modified antibody having a structure of uncharged hydrophilic polymer block-environmentally responsive bond-antibody is provided. Here, the symbol "-" represents a bond or spacer, and means that the element described before the symbol and the element described after the symbol are linked via a bond or spacer. Spacers have chemical properties that are stable in vivo. In certain aspects of the invention, the eco-responsive bond is a reduced eco-responsive bond, eg, a disulfide bond. In the present invention, the GLUT1 ligand can be glucose. Glucose is linked to the uncharged hydrophilic polymer block so that it can bind to GLUT1.
　本発明によれば、
－Ｃ（Ｏ）－Ｏ－Ｌ1－Ｓ－Ｓ－Ｌ2－（非電荷親水性ポリマーブロック）により修飾されたアミノ基を有する、抗体が提供される。Ｌ1は置換されていてもよい低級アルキレン（すなわち、置換または非置換低級アルキレン）であり、好ましくはエチレンである。Ｌ2は、結合（単結合）、または非開裂型のスペーサー（例えば、安定なスペーサー）である。非電荷親水性ポリマーブロックは、標的化分子によりさらに修飾されていてもよいし、標的化分子により修飾されていなくてもよい。この態様において、Ｌ2は、Ｌ3－Ｏ－Ｃ（Ｏ）－ＮＨ－Ｌ4であり得る。このような抗体は、還元環境下において、ジスルフィド結合において開裂すると、未修飾型の抗体が放出され得る。本発明によれば、放出された未修飾型の抗体は、抗原への本来の結合親和性を回復し得る。 According to the invention,
Antibodies are provided having amino groups modified with -C(O)-OL 1 -SSL 2 - (uncharged hydrophilic polymer blocks). L 1 is optionally substituted lower alkylene (that is, substituted or unsubstituted lower alkylene), preferably ethylene. L2 is a bond ( single bond) or a non-cleavable spacer (eg, a stable spacer). The uncharged hydrophilic polymer block may or may not be further modified with a targeting molecule. In this aspect, L 2 can be L 3 —O—C(O)—NH—L 4 . Such antibodies can be cleaved at the disulfide bonds in a reducing environment to release the unmodified form of the antibody. According to the invention, the released unmodified antibody can regain its original binding affinity for the antigen.
　本発明によれば、
－Ｃ（Ｏ）－Ｏ－Ｌ1－Ｓ－Ｓ－Ｌ3－Ｏ－Ｃ（Ｏ）－ＮＨ－Ｌ4－（非電荷親水性ポリマーブロック）
｛式中、
　Ｌ1、Ｌ3およびＬ4は、それぞれ独立して、結合または置換されていてもよい低級アルキレンである｝により修飾されたアミノ基を有する、抗体
が提供される。この抗体は、還元環境応答性再活性化可能抗体（例えば、脳標的化型再活性化可能抗体）であり得る。非電荷親水性ポリマーブロックは、標的化分子によりさらに修飾されていてもよいし、標的化分子により修飾されていなくてもよい。本発明では、標的化分子は、ＧＬＵＴ１リガンドであり得る。本発明では、ＧＬＵＴ１リガンドは、グルコースであり得る。グルコースは、ＧＬＵＴ１と結合可能に、非電荷親水性ポリマーブロックと連結している。このような抗体は、還元環境下において、ジスルフィド結合において開裂すると、未修飾型の抗体が放出され得る。本発明によれば、放出された未修飾型の抗体は、抗原への本来の結合親和性を回復し得る。 According to the invention,
-C(O)-OL 1 -SSL 3 -OC(O)-NH-L 4 - (uncharged hydrophilic polymer block)
{In the formula,
L 1 , L 3 and L 4 are each independently a bond or optionally substituted lower alkylene}. The antibody can be a reducing environment-responsive reactivatable antibody (eg, a brain-targeted reactivatable antibody). The uncharged hydrophilic polymer block may or may not be further modified with a targeting molecule. In the present invention the targeting molecule can be a GLUT1 ligand. In the present invention, the GLUT1 ligand can be glucose. Glucose is linked to the uncharged hydrophilic polymer block so that it can bind to GLUT1. Such antibodies can be cleaved at the disulfide bonds in a reducing environment to release the unmodified form of the antibody. According to the invention, the released unmodified antibody can regain its original binding affinity for the antigen.
　上記抗体は、環境応答性に、例えば、還元環境（例えば、脳実質または腫瘍組織の還元環境）下、または低ｐH条件（例えば、生理学的に可能な低ｐH条件）下でジスルフィド結合（－Ｓ－Ｓ－）において開裂する。ジスルフィド結合が開裂すると、非電荷親水性ポリマーブロックが抗体から乖離し、抗体は、抗原に対する結合親和性を回復させ得る。 The antibodies are environmentally responsive, e.g., disulfide bonds (-S -S-). Cleavage of the disulfide bond releases the uncharged hydrophilic polymer block from the antibody, allowing the antibody to regain binding affinity for the antigen.
　ある態様では、上記抗体は、ジスルフィド結合開裂後、連鎖反応により、インタクトな抗体（または未修飾型の抗体または天然型の抗体）を放出し得る。インタクトな抗体とは、修飾を受けていたアミノ基が、－ＮＨ2基に戻ることを意味する。放出されたインタクトな抗体は、元々の抗体の抗原に対する結合親和性を回復させ得る。 In some embodiments, the antibody can release an intact antibody (or unmodified or native antibody) by chain reaction after disulfide bond cleavage. An intact antibody means that the modified amino groups have reverted to --NH 2 groups. The released intact antibody can restore the original antibody's binding affinity for the antigen.
　ある態様では、抗体は、非電荷親水性ポリマーブロックによる修飾によって、その抗原に対する結合親和性を減少または喪失している。ある態様では、抗体は、非電荷親水性ポリマーブロックによる修飾によって、その抗原に対する結合親和性を５０％以下、４０％以下、３０％以下、２０％以下、１０％以下、５％以下、４％以下、３％以下、２％以下、１％以下、０．１％以下、または０．０１％以下にまで減少させている。抗体は、非電荷親水性ポリマーブロックによる修飾によって、結合親和性を減少または喪失すると、環境応答性の結合が開裂するまでは不活性であり、それ故に、標的化部位以外で抗体が機能することを防ぐことができる。このようにすると、抗体の機能を標的部位に特異的に回復させることができるようになる。 In some embodiments, the antibody has reduced or lost binding affinity for its antigen by modification with an uncharged hydrophilic polymer block. In some embodiments, the antibody has a binding affinity for its antigen that is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 4% or less by modification with an uncharged hydrophilic polymer block. Below, it is reduced to 3% or less, 2% or less, 1% or less, 0.1% or less, or 0.01% or less. Antibodies that have reduced or lost binding affinity by modification with uncharged hydrophilic polymer blocks are inactive until the environmentally responsive bond is cleaved, thus functioning the antibody outside of the targeted site. can be prevented. In this way, the function of the antibody can be restored specifically to the target site.
　抗体は、ジスルフィド結合が開裂する環境、例えば、還元環境（例えば、脳実質または腫瘍組織の還元環境）、または低ｐH条件（例えば、生理学的に可能な低ｐH条件）に存在する抗原に結合しうる。未修飾型の抗体において、抗原に対する結合親和性（ＫＤ）は、例えば、１０－８Ｍ以下、１０－９Ｍ以下、１０－１０Ｍ以下、１０－１１Ｍ以下、または１０－１２Ｍ以下であり得る。 Antibodies bind antigens in an environment where disulfide bonds are cleaved, e.g., in a reducing environment (e.g., the reducing environment of brain parenchyma or tumor tissue), or in low pH conditions (e.g., physiologically possible low pH conditions). sell. In unmodified antibodies, the binding affinity (KD) for antigen is, for example, 10 −8 M or less, 10 −9 M or less, 10 −10 M or less, 10 −11 M or less, or 10 −12 M or less. could be.
　ジスルフィド結合が開裂する環境、例えば、還元環境（例えば、脳実質または腫瘍組織の還元環境）、または低ｐH条件（例えば、生理学的に可能な低ｐH条件）に存在する抗原は、例えば、がん抗原、または免疫チェックポイント分子であり得る。免疫チェックポイント分子とは、免疫チェックポイントに関与する分子を意味する。免疫チェックポイント分子は、そのカウンターパート（すなわち、結合相手である免疫チェックポイント分子）と結合して、免疫チェックポイントを活性化させ、免疫作用を抑制する。免疫チェックポイント分子としては、例えば、ＰＤ－１系免疫チェックポイントに関与する分子であり得る。ＰＤ－１系免疫チェックポイントに関与する分子としては、プログラム細胞死－１（ＰＤ－１）、細胞傷害性Ｔリンパ球関連タンパク質４（ｃｙｔｏｔｏｘｉｃ　Ｔ－ｌｙｍｐｈｏｃｙｔｅ－ａｓｓｏｃｉａｔｅｄ　ｐｒｏｔｅｉｎ　４；ＣＴＬＡ－４）、Ｔ細胞免疫グロブリンドメインおよびムチンドメイン－３（Ｔ－ｃｅｌｌ　ｉｍｍｕｎｏｇｌｏｂｕｌｉｎ　ｄｏｍａｉｎ　ａｎｄ　ｍｕｃｉｎ　ｄｏｍａｉｎ－３、またはＴＩＭ－３）、リンパ球活性化遺伝子３（ｌｙｍｐｈｏｃｙｔｅ　ａｃｔｉｖａｔｉｏｎ　ｇｅｎｅ　３、またはＬＡＧ－３）、およびＶ型免疫グロブリンドメイン含有Ｔ細胞活性化抑制因子（Ｖ－ｔｙｐｅ　ｉｍｍｕｎｏｇｌｏｂｕｌｉｎ　ｄｏｍａｉｎ－ｃｏｎｔａｉｎｉｎｇ　ｓｕｐｐｒｅｓｓｏｒ　ｏｆ　Ｔ－ｃｅｌｌ　ａｃｔｉｖａｔｉｏｎ、またはＶＩＳＴＡ）が挙げられる。それぞれが担う免疫チェックポイントはＰＤ－１系免疫チェックポイント、ＣＴＬＡ－４系免疫チェックポイント、ＴＩＭ－３系免疫チェックポイント、ＬＡＧ－３系免疫チェックポイント、およびＶＩＳＴＡ系免疫チェックポイントとよばれる。免疫チェックポイント阻害剤は、例えば免疫チェックポイント分子又はそのカウンターパートに結合して、免疫チェックポイントの機能を阻害し得る。例えば、ＰＤ－１とＰＤ－Ｌ１又はＰＤ－Ｌ２との結合を阻害することにより、ＰＤ－１系免疫チェックポイントを阻害することができる。また、ＣＴＬＡ－４とＣＤ８０又はＣＤ８６との結合を阻害することにより、ＣＴＬＡ－４系免疫チェックポイントを阻害することができる。また、ＴＩＭ－３とガレクチン－９との結合を阻害することにより、ＴＩＭ－３系免疫チェックポイントを阻害することができる。また、ＬＡＧ－３とＭＨＣクラスＩＩ分子との結合を阻害することにより、ＬＡＧ－３系免疫チェックポイントを阻害することができる。また、ＶＩＳＴＡとＶＳＩＧ－３／ＩＧＳＦ１１との結合を阻害することにより、ＶＩＳＴＡ系免疫チェックポイントを阻害することができる。このようにして、ＰＤ－１系免疫チェックポイント、ＣＴＬＡ－４系免疫チェックポイント、ＴＩＭ－３系免疫チェックポイント、Ｌａｇ３系免疫チェックポイント、およびＶＩＳＴＡ系免疫チェックポイントからなる群から選択される１以上の免疫チェックポイントを阻害することができる。２つのタンパク質の結合を阻害する抗体は、免疫チェックポイント分子またはそのカウンターパートに結合することができる。例えば、ＰＤ－１系免疫チェックポイントを阻害する抗体は、抗ＰＤ－１抗体、抗ＰＤ－Ｌ１抗体、および抗ＰＤ－Ｌ２抗体からなる群から選択される抗体（例えば、ニボルマブ、ペムプロリズマブ、アベルマブ、アテゾリズマブ、およびヂュルバルマブ）であり得る。また、ＣＴＬＡ－４系免疫チェックポイントを阻害する抗体は、抗ＣＤＬＡ－４抗体、抗ＣＤ８０抗体、および抗ＣＤ８６抗体からなる群から選択される抗体（例えば、イピリムマブ、およびトレメリムマブ）であり得る。また、ＴＩＭ－３系免疫チェックポイントを阻害する抗体は、抗ＴＩＭ－３抗体および抗ガレクチン－９抗体からなる群から選択される抗体（例えば、ＭＧＢ４５３）であり得る。また、また、ＶＩＳＴＡ系免疫チェックポイントを阻害する抗体は、抗ＶＩＳＴＡ抗体および抗ＶＳＩＧ－３／ＩＧＳＦ１１抗体からなる群から選択される抗体（例えば、ＪＮＪ－６１６１０５８８）であり得る。
　ある好ましい態様では、本発明に用いられる抗体は、免疫細胞に発現する免疫チェックポイント分子（例えば、ＰＤ－１、ＣＴＬＡ４、ＬＡＧ－３、ＴＩＧＩＴ、ＶＩＳＴＡおよびＴＩＭ－３）に結合する。本発明の別の好ましい態様では、本発明で用いられる抗体は、免疫細胞に発現する免疫チェックポイント分子のカウンターパート（例えば、ＰＤ－Ｌ１、ＰＤ－Ｌ２、ＣＤ８０、ＣＤ８６、ガレクチン－９、ＭＨＣクラスＩＩ分子、ＶＳＩＧ－３／ＩＧＳＦ１１）に結合する。
　ＰＤ－１系免疫チェックポイントは、ＰＤ－１およびＰＤ－Ｌ１の結合を遮断することによって阻害することができる。または、ＰＤ－１系免疫チェックポイントは、ＰＤ－１シグナルを遮断することによって阻害することができる。
　したがって、本発明の抗体は、免疫チェックポイント阻害剤であり得る。本発明の抗体は、免疫チェックポイント分子のいずれかに結合し、そのカウンターパートとなる免疫チェックポイント分子との結合を遮断することができる。本発明の抗体は、ＰＤ－１系免疫チェックポイント阻害剤であり得る。本発明の抗体は、例えば、ＰＤ－１およびＰＤ－Ｌ１の結合を遮断することができる抗体であり得る。本発明の抗体は、例えば、ＰＤ－１に結合する抗体（または、抗ＰＤ－１抗体）であり得る。本発明の抗体は、例えば、ＰＤ－Ｌ１に結合する抗体（または、抗ＰＤ－Ｌ１抗体）であり得る。本発明の抗体は、ＰＤ－１およびＰＤ－Ｌ１の結合を遮断することができる抗ＰＤ－Ｌ１抗体であり得る。本発明の抗体は、例えば、ＰＤ－１およびＰＤ－Ｌ１の結合を遮断することができる抗ＰＤ－１抗体であり得る。遮断とは、結合を５０％以上、６０％以上、７０％以上、８０％以上、９０％以上、または９５％以上阻害することを意味し得る。 Antigens present in environments where disulfide bonds are cleaved, e.g., in reducing environments (e.g., the reducing environment of brain parenchyma or tumor tissue), or in low pH conditions (e.g., physiologically possible low pH conditions), are associated with cancers, e.g. It can be an antigen, or an immune checkpoint molecule. An immune checkpoint molecule means a molecule involved in an immune checkpoint. Immune checkpoint molecules bind to their counterparts (ie, binding partners immune checkpoint molecules) to activate immune checkpoints and suppress immune action. The immune checkpoint molecule can be, for example, a molecule involved in the PD-1 system immune checkpoint. Molecules involved in the PD-1 immune checkpoint include programmed cell death-1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T Cellular immunoglobulin domain and mucin domain-3 (T-cell immunoglobulin domain and mucin domain-3, or TIM-3), lymphocyte activation gene 3 (LAG-3), and type V immunoglobulin domain-containing suppressor of T-cell activation (V-type immunoglobulin domain-containing suppressor of T-cell activation, or VISTA). The immune checkpoints responsible for each are called PD-1 immune checkpoint, CTLA-4 immune checkpoint, TIM-3 immune checkpoint, LAG-3 immune checkpoint, and VISTA immune checkpoint. An immune checkpoint inhibitor can inhibit the function of an immune checkpoint, eg, by binding to an immune checkpoint molecule or its counterpart. For example, by inhibiting the binding of PD-1 to PD-L1 or PD-L2, the PD-1 system immune checkpoint can be inhibited. Also, by inhibiting the binding of CTLA-4 to CD80 or CD86, the CTLA-4 system immune checkpoint can be inhibited. In addition, by inhibiting the binding of TIM-3 to galectin-9, the TIM-3 system immune checkpoint can be inhibited. In addition, by inhibiting the binding of LAG-3 to MHC class II molecules, the LAG-3 system immune checkpoint can be inhibited. Also, by inhibiting the binding of VISTA and VSIG-3/IGSF11, the VISTA system immune checkpoint can be inhibited. Thus, one or more selected from the group consisting of PD-1-based immune checkpoint, CTLA-4-based immune checkpoint, TIM-3-based immune checkpoint, Lag3-based immune checkpoint, and VISTA-based immune checkpoint can inhibit the immune checkpoint of Antibodies that inhibit the binding of two proteins can bind to immune checkpoint molecules or their counterparts. For example, antibodies that inhibit the PD-1 system immune checkpoint are antibodies selected from the group consisting of anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies (e.g., nivolumab, pemprolizumab, avelumab, atezolizumab, and durvalumab). Antibodies that inhibit CTLA-4-based immune checkpoints can also be antibodies selected from the group consisting of anti-CDLA-4 antibodies, anti-CD80 antibodies, and anti-CD86 antibodies (eg, ipilimumab and tremelimumab). Also, the antibody that inhibits the TIM-3-based immune checkpoint can be an antibody (eg, MGB453) selected from the group consisting of anti-TIM-3 antibodies and anti-galectin-9 antibodies. Also, the antibody that inhibits the VISTA-based immune checkpoint can be an antibody (eg, JNJ-61610588) selected from the group consisting of anti-VISTA antibody and anti-VSIG-3/IGSF11 antibody.
In one preferred embodiment, the antibodies used in the present invention bind to immune checkpoint molecules (eg, PD-1, CTLA4, LAG-3, TIGIT, VISTA and TIM-3) expressed on immune cells. In another preferred embodiment of the present invention, the antibodies used in the present invention are immune checkpoint molecule counterparts expressed on immune cells (e.g., PD-L1, PD-L2, CD80, CD86, galectin-9, MHC class II molecule, VSIG-3/IGSF11).
The PD-1 based immune checkpoint can be inhibited by blocking the binding of PD-1 and PD-L1. Alternatively, the PD-1 system immune checkpoint can be inhibited by blocking the PD-1 signal.
Accordingly, the antibodies of the invention can be immune checkpoint inhibitors. Antibodies of the invention can bind to any of the immune checkpoint molecules and block binding to their counterpart immune checkpoint molecules. Antibodies of the invention can be PD-1 system immune checkpoint inhibitors. An antibody of the invention can be, for example, an antibody capable of blocking the binding of PD-1 and PD-L1. An antibody of the invention can be, for example, an antibody that binds to PD-1 (or an anti-PD-1 antibody). An antibody of the invention can be, for example, an antibody that binds to PD-L1 (or an anti-PD-L1 antibody). Antibodies of the invention can be anti-PD-L1 antibodies capable of blocking the binding of PD-1 and PD-L1. Antibodies of the invention can be, for example, anti-PD-1 antibodies capable of blocking the binding of PD-1 and PD-L1. Blocking can mean inhibiting binding by 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more.
　血流で安定なスペーサーは、血流で投与から標的組織（例えば、脳実質または腫瘍組織）への移行に必要な時間にわたって安定に血中で存在し得る程度に安定であるスペーサーを意味する。炭素間結合、アミド結合、ホスホジエステル結合、エステル結合、エーテル結合、アルキレン、カーバメート結合、チオカーバメート結合、チオエステル結合、チオエーテル結合、およびジスルフィド結合並びにこれらの組合せからなる群から選択される結合は、血流で安定なスペーサーであり得る。本発明では、血流で安定なスペーサーは、薬学的に許容可能である。従って、非電荷親水性ポリマーセグメントと抗体とは、これらのスペーサーによって連結され得る。血流で安定なスペーサーとしては、特に限定されないが例えば、置換若しくは非置換アルキレン、または置換若しくは非置換ヘテロアルキレンである。スペーサーが血流で安定かどうかは、例えば、単離された血液、または血清を含む生理食塩水中で、スペーサーの安定性を評価することによって決定することができる。投与から標的組織（例えば、脳実質または腫瘍組織）への移行に必要な時間は、当業者であれば適宜決定することができる。投与から標的組織（例えば、脳実質または腫瘍組織）への移行に必要な時間は、例えば、１時間以上、２時間以上、３時間以上、４時間以上、５時間以上、６時間以上、７時間以上、８時間以上、９時間以上、１０時間以上、１２時間以上、１５時間以上、１８時間以上、２１時間以上、２４時間以上、２日以上、３日以上、４日以上、５日以上、６日以上、または７日以上であり得る。投与から標的組織（例えば、脳実質または腫瘍組織）への移行に必要な時間は、例えば、7日以下、6日以下、5日以下、4日以下、3日以下、2日以下、または1日以下であり得る。投与から標的組織（例えば、脳実質または腫瘍組織）への移行に必要な時間は、例えば、1時間以上～1日以下であり得る。 A bloodstream-stable spacer means a spacer that is stable to the extent that it can be stably present in the blood over the time required for its transition from administration to target tissue (for example, brain parenchyma or tumor tissue) in the bloodstream. A bond selected from the group consisting of a carbon-carbon bond, an amide bond, a phosphodiester bond, an ester bond, an ether bond, an alkylene, a carbamate bond, a thiocarbamate bond, a thioester bond, a thioether bond, a disulfide bond, and combinations thereof It can be a flow-stable spacer. In the present invention, a bloodstream stable spacer is pharmaceutically acceptable. Therefore, the uncharged hydrophilic polymer segment and the antibody can be linked by these spacers. Examples of spacers that are stable in the bloodstream include, but are not limited to, substituted or unsubstituted alkylene or substituted or unsubstituted heteroalkylene. Whether a spacer is stable in the blood stream can be determined by assessing the stability of the spacer in, for example, isolated blood or serum-containing saline. A person skilled in the art can appropriately determine the time required from administration to target tissue (eg, brain parenchyma or tumor tissue). The time required for the transition from administration to the target tissue (e.g., brain parenchyma or tumor tissue) is, for example, 1 hour or longer, 2 hours or longer, 3 hours or longer, 4 hours or longer, 5 hours or longer, 6 hours or longer, and 7 hours. 8 hours or more, 9 hours or more, 10 hours or more, 12 hours or more, 15 hours or more, 18 hours or more, 21 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, It can be 6 days or more, or 7 days or more. The time required to transition from administration to the target tissue (e.g., brain parenchyma or tumor tissue) is, for example, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less. can be days or less. The time required for transit from administration to target tissue (eg, brain parenchyma or tumor tissue) can be, for example, from 1 hour or more to 1 day or less.
　本発明によれば、非電荷親水性ポリマーセグメントで修飾された抗体であって、抗体は、抗原に結合することができ、血中では、非電荷親水性ポリマーセグメントで修飾された抗体の形態であり、血管内からジスルフィド結合が開裂する特別な環境下（例えば、脳実質または腫瘍組織）に移行した後に、抗体は、非電荷親水性ポリマーセグメントと解離した形態である、抗体が提供される。 According to the present invention, an antibody modified with uncharged hydrophilic polymer segments, the antibody being able to bind to an antigen, in blood in the form of an antibody modified with uncharged hydrophilic polymer segments The antibody is provided in a form dissociated from the uncharged hydrophilic polymer segment after being transferred from the blood vessel to a special environment where disulfide bonds are cleaved (e.g., brain parenchyma or tumor tissue).
　本発明のある態様によれば、上記抗体は、血管内皮細胞を通過してジスルフィド結合が開裂する特別な環境に入ると、非電荷親水性ポリマーセグメントと解離する。例えば、上記抗体は、腫瘍組織に入ると、腫瘍組織内で、または脳実質に入ると、ＢＢＢのトランスサイトーシス中に、もしくは脳実質で、非電荷親水性ポリマーセグメントと解離する。この態様では、上記抗体は、非電荷親水性ポリマーセグメントと、還元環境応答性のリンカーで連結されており、当該結合は標的組織（例えば、脳実質または腫瘍組織）内の還元環境下で開裂する。この態様において、還元環境応答性の結合は、ジスルフィド結合であり得る。
　本発明のある態様によれば、非電荷親水性ポリマーセグメントと解離した抗体は、非電荷親水性ポリマーセグメントで修飾された抗体よりも、抗原への結合親和性が強い。
　本発明にある態様によれば、非電荷親水性ポリマーセグメントで修飾された抗体は、修飾前の抗体（未修飾の抗体）よりも抗原への結合親和性が弱い。
　本発明のある態様によれば、非電荷親水性ポリマーセグメントで修飾された抗体は、修飾前の抗体（未修飾の抗体）よりも抗原への結合親和性が弱く、かつ、非電荷親水性ポリマーセグメントと解離した抗体は、非電荷親水性ポリマーセグメントで修飾された抗体よりも、抗原への結合親和性が強い。
　本発明によれば、非電荷親水性ポリマーセグメントで修飾された抗体は、ジスルフィド結合が開裂する特別な環境、例えば、脳実質または腫瘍組織内では、前記セグメントと開裂しており、修飾によって低下していた抗体の結合特性を回復させることができる。このような抗体は、例えば、血管内皮細胞が脆弱な、または血液脳関門が脆弱化した疾患の患者においては、特別な操作（例えば、血糖操作）をしなくても、また、抗体へのグルコースによる修飾をしなくても、当該環境（例えば、脳実質または腫瘍組織）に到達し得、当該環境（脳実質または腫瘍組織）内でその結合特性を回復させることから有用であり得る。本発明によれば、この態様において前記非電荷親水性ポリマーセグメントは、ＧＬＵＴ１リガンドで修飾されていてもよい。ＧＬＵＴ１リガンドによる修飾は、ＧＬＵＴ１を発現する血管内皮（例えば、血液脳関門）を有する対象において、当該抗体を血管内皮細胞（例えば、脳血管内皮細胞）のエンドソーム内または標的組織（例えば、脳実質または腫瘍組織）内に送る観点で有用である。 According to one aspect of the invention, the antibody dissociates from the uncharged hydrophilic polymer segments upon passage through vascular endothelial cells into a special environment where disulfide bonds are cleaved. For example, the antibody dissociates with uncharged hydrophilic polymer segments upon entering tumor tissue, within tumor tissue, or upon entering the brain parenchyma, during transcytosis of the BBB, or in the brain parenchyma. In this aspect, the antibody is linked to an uncharged hydrophilic polymer segment with a reducing environment-responsive linker, which bond is cleaved under a reducing environment within the target tissue (e.g., brain parenchyma or tumor tissue). . In this aspect, the reducing environment-responsive bond can be a disulfide bond.
According to one aspect of the invention, antibodies dissociated with uncharged hydrophilic polymer segments have a stronger binding affinity for antigen than antibodies modified with uncharged hydrophilic polymer segments.
According to one aspect of the invention, the antibody modified with uncharged hydrophilic polymer segments has a weaker binding affinity to antigen than the antibody before modification (unmodified antibody).
According to one aspect of the present invention, an antibody modified with a non-charged hydrophilic polymer segment has a weaker binding affinity to an antigen than an antibody before modification (unmodified antibody), and the non-charged hydrophilic polymer Antibodies with dissociated segments have a stronger binding affinity for antigen than antibodies modified with uncharged hydrophilic polymer segments.
According to the present invention, antibodies modified with uncharged hydrophilic polymer segments are cleaved with said segments in special environments where disulfide bonds are cleaved, e.g. within brain parenchyma or tumor tissue, and the modification reduces It can restore the binding properties of the antibody that had been lost. Such antibodies can be used without special manipulations (e.g., glycemic manipulation) in patients with diseases in which vascular endothelial cells are weakened or the blood-brain barrier is weakened. It can be useful because it can reach the environment (eg, brain parenchyma or tumor tissue) and restore its binding properties within the environment (brain parenchyma or tumor tissue) without modification by . According to the invention, in this aspect said uncharged hydrophilic polymer segment may be modified with a GLUT1 ligand. Modification with a GLUT1 ligand can target the antibody within endosomes of vascular endothelial cells (e.g., brain vascular endothelial cells) or in target tissues (e.g., brain parenchyma or It is useful from the viewpoint of sending it into the tumor tissue).
　本発明によれば、非電荷親水性ポリマーによってリンカーを介して修飾された抗体であって、非電荷親水性ポリマーがＧＬＵＴ１リガンドで修飾された抗体が提供される。当該抗体は、腫瘍組織の血管を通り抜け、ＥＰＲ効果等により腫瘍組織内へ進入することができる。あるいは、当該抗体は、血管内皮細胞（例えば、脳血管内皮細胞）の管腔側表面に発現したＧＬＵＴ１と、ＧＬＵＴ１リガンドを介して結合することができる。血糖を低下させた対象に対して、グルコースを投与（またはＧＬＵＴ１リガンドを投与）すると、当該対象の血管内皮細胞（例えば、脳血管内皮細胞）の管腔側表面に結合した物質はエンドサイトーシスによって血管内皮細胞内に取り込まれ、その少なくとも一部は、トランスサイトーシスによってジスルフィド結合が開裂する特別な環境（例えば、脳実質）内に送達される。血糖を低下させた対象の血管内皮細胞（例えば、脳血管内皮細胞）の管腔側表面にはＧＬＵＴ１が発現しており、本発明の上記抗体は、ＧＬＵＴ１リガンドを介してＧＬＵＴ１に結合し、グルコースを投与（またはＧＬＵＴ１リガンドを投与）すると、エンドサイトーシスによって血管内皮細胞内に取り込まれ、その少なくとも一部は、トランスサイトーシスによって上記環境（例えば、脳実質）内に送達される。ここで、ＧＬＵＴ１と当該抗体との結合は、インビトロ実験において、単離したＧＬＵＴ１と当該抗体との結合を評価するアッセイによって確認することができる。非電荷親水性ポリマーとしては、例えば、ポリエチレングリコール（ＰＥＧ）、およびポリオキサゾリンなどの非電荷親水性のポリマーが挙げられる。ここで、「非電荷」とは、当該ポリマーセグメント全体において、電荷が中和していることを意味する。非電荷親水性ポリマーは、生体適合性である。
　ある態様では、本発明の抗体は、上記ＧＬＵＴ１リガンドとは別の標的組織（例えば、脳実質または腫瘍組織）内の受容体に対するリガンド（第２のリガンド）で修飾された非電荷親水性ポリマーセグメントを有することができ、または、有しないことができる。 According to the present invention, there is provided an antibody modified with an uncharged hydrophilic polymer via a linker, wherein the uncharged hydrophilic polymer is modified with a GLUT1 ligand. The antibody can pass through the blood vessels of tumor tissue and enter into tumor tissue due to the EPR effect or the like. Alternatively, the antibody can bind to GLUT1 expressed on the luminal surface of vascular endothelial cells (eg, cerebral vascular endothelial cells) via GLUT1 ligands. When glucose is administered (or GLUT1 ligand is administered) to a hypoglycemic subject, substances bound to the luminal surface of vascular endothelial cells (e.g., cerebral vascular endothelial cells) of the subject undergo endocytosis. It is taken up into vascular endothelial cells and at least part of it is delivered by transcytosis into a special environment (eg brain parenchyma) where disulfide bonds are cleaved. GLUT1 is expressed on the luminal surface of vascular endothelial cells (e.g., cerebral vascular endothelial cells) of a subject whose blood sugar is lowered, and the antibody of the present invention binds to GLUT1 via a GLUT1 ligand, (or a GLUT1 ligand) is taken up by endocytosis into vascular endothelial cells, at least a portion of which is delivered by transcytosis into the environment (eg, brain parenchyma). Here, the binding of GLUT1 to the antibody can be confirmed by an assay that evaluates the binding of isolated GLUT1 to the antibody in an in vitro experiment. Uncharged hydrophilic polymers include, for example, polyethylene glycol (PEG) and uncharged hydrophilic polymers such as polyoxazolines. Here, "uncharged" means that the charge is neutralized throughout the polymer segment. Uncharged hydrophilic polymers are biocompatible.
In one aspect, the antibody of the invention is an uncharged hydrophilic polymer segment modified with a ligand (second ligand) for a receptor in a target tissue (e.g., brain parenchyma or tumor tissue) other than the GLUT1 ligand or not.
　本発明によれば、本発明の上記抗体においてリンカーは、抗体のリジン残基の側鎖アミノ基に連結させることができる。当該連結は、好ましくは、共有結合であり得る。ある態様では、リンカーを連結したリジン残基は、抗体の重鎖および／または軽鎖可変領域内に存在し得る。ある態様では、リンカーを連結したリジン残基は、抗体の重鎖および／または軽鎖可変領域のＣＤＲ領域内に存在し得る。
　標的化分子（例えば、ＧＬＵＴ１リガンド）による修飾は、血流からジスルフィド結合が開裂する特別な環境（例えば、脳実質または腫瘍組織）に移行するために用いられ、ジスルフィド結合が開裂する特別な環境（例えば、脳実質または腫瘍組織）に移行した後に不要となり得る。従って、抗体がジスルフィド結合が開裂する特別な環境（例えば、脳実質または腫瘍組織）に移行した後には、抗体を修飾する標的化分子（例えば、ＧＬＵＴ１リガンド）により修飾されたＰＥＧは、抗体から切り離されてもよい。また、本発明によれば、抗体のリジン残基の側鎖アミノ基の修飾は、修飾の強度によっては抗体の結合特性を低下させ得る。従って、抗体がジスルフィド結合が開裂する特別な環境（例えば、脳実質または腫瘍組織）に移行した後には、抗体を修飾する標的化分子（例えば、ＧＬＵＴ１リガンド)により修飾されたＰＥＧは、抗体から切り離されてもよい。標的化分子（例えば、ＧＬＵＴ１リガンド）は、ＰＥＧを修飾し得る。標的化分子（例えば、ＧＬＵＴ１リガンド）は、ＰＥＧの末端の炭素原子または酸素原子を修飾し得る。
　ジスルフィド結合が開裂する特別な環境は、例えば、脳実質または腫瘍組織であり得る。例えば、脳の実質または腫瘍組織は、還元環境を有する。脳実質または腫瘍組織の還元環境は、２ｍＭのグルタチオン（ＧＳＨ）水溶液と同等の強度の還元環境である。リンカー中に、還元環境下で開裂することができる開裂部位を含めておくことによって、抗体が脳実質または腫瘍組織に送達された後に、リンカーが切断されるようにリンカーを構成することができる。このようなリンカーを還元環境下において開裂可能なリンカーという。本発明では、標的化分子（例えば、ＧＬＵＴ１リガンド）により修飾された非電荷親水性ポリマー（例えば、ＰＥＧ）によって還元環境下において開裂可能なリンカーを介して修飾された抗体が提供される。本発明の抗体は、好ましくは、還元的環境を提供する脳実質または腫瘍組織においてリンカーを開裂させ得る。還元環境下において開裂可能なリンカーは、例えば、開裂部位としてジスルフィド結合を有し得る。還元環境下において開裂可能なリンカーを用いて、抗体と標的化分子（例えば、ＧＬＵＴ１リガンド）により修飾されたＰＥＧを連結することによって、抗体が脳実質または腫瘍組織に送達された後に、リンカーが切断され、脳実質または腫瘍組織内で抗体を放出させることができる。 According to the present invention, the linkers in the above antibodies of the present invention can be linked to side chain amino groups of lysine residues of the antibody. The linkage may preferably be a covalent bond. In certain aspects, the lysine residues that join the linkers can be present in the heavy and/or light chain variable regions of the antibody. In certain aspects, the lysine residues that join the linkers can be present within the CDR regions of the heavy and/or light chain variable regions of the antibody.
Modification with a targeting molecule (e.g. GLUT1 ligand) is used to translocate from the bloodstream to a special environment (e.g. brain parenchyma or tumor tissue) where disulfide bonds are cleaved (e.g. brain parenchyma or tumor tissue). for example, brain parenchyma or tumor tissue). Therefore, after the antibody is transferred to a special environment where the disulfide bond is cleaved (e.g., brain parenchyma or tumor tissue), the PEG modified by the antibody-modifying targeting molecule (e.g., GLUT1 ligand) is cleaved off from the antibody. may be Also, according to the present invention, modification of the side chain amino groups of lysine residues of antibodies can reduce the binding properties of antibodies depending on the strength of the modification. Therefore, after the antibody is transferred to a special environment where disulfide bonds are cleaved (e.g., brain parenchyma or tumor tissue), the PEG modified by the antibody-modifying targeting molecule (e.g., GLUT1 ligand) is cleaved off from the antibody. may be A targeting molecule (eg, a GLUT1 ligand) may modify PEG. A targeting molecule (eg, a GLUT1 ligand) can modify a terminal carbon or oxygen atom of PEG.
A particular environment in which disulfide bonds are cleaved can be, for example, brain parenchyma or tumor tissue. For example, brain parenchyma or tumor tissue has a reducing environment. The reducing environment of brain parenchyma or tumor tissue is a reducing environment of strength equivalent to 2 mM glutathione (GSH) aqueous solution. The linker can be configured such that the linker is cleaved after the antibody is delivered to the brain parenchyma or tumor tissue by including a cleavage site in the linker that can be cleaved under a reducing environment. Such linkers are referred to as cleavable linkers in a reducing environment. The present invention provides antibodies modified by an uncharged hydrophilic polymer (eg, PEG) modified with a targeting molecule (eg, a GLUT1 ligand) via a linker that is cleavable in a reducing environment. Antibodies of the invention are preferably capable of cleaving the linker in brain parenchyma or tumor tissue that provides a reducing environment. A linker that is cleavable under a reducing environment can, for example, have a disulfide bond as the cleavage site. By linking an antibody and a targeting molecule (e.g., GLUT1 ligand)-modified PEG with a linker that is cleavable under a reducing environment, the linker is cleaved after the antibody has been delivered to the brain parenchyma or tumor tissue. and can release antibodies within the brain parenchyma or tumor tissue.
　別の態様では、還元環境下において開裂可能なリンカーは、開裂によって抗体のリジン残基の側鎖アミノ基を非置換のアミノ基となるように構成することができる。例えば、リンカーとして、（抗体－ＮＨ）－ＣＯ－Ｏ－Ｃ２Ｈ４－Ｓ－Ｓ－Ｌ２－（非電荷親水性ポリマー）｛ここで、Ｌ２は、結合、または血流で安定なリンカーである｝を用いた場合には、リンカー中のジスルフィド結合が開裂すると以下のような反応によって、抗体のリジン残基の側鎖アミノ基を回復させる。その結果、上記修飾抗体は、未修飾型抗体（インタクトな抗体）を放出する。以下では、ＧＬＵＴ１リガンドがグルコース（Ｇｌｕｃ）であり、非電荷親水性ポリマーがポリエチレングリコール（ＰＥＧ）であり、Ｌ２が－Ｃ２Ｈ４－Ｏ－ＣＯ－である本発明の抗体が還元環境下でリンカーと解離して元の抗体に戻る機構が示される。この機構は、ＧＬＵＴ１リガンドがグルコース以外のＧＬＵＴ１リガンドであり、非電荷親水性ポリマーがＰＥＧ以外の非電荷親水性ポリマーである場合も同様である。 In another aspect, a linker that is cleavable in a reducing environment can be configured such that cleavage results in side chain amino groups of lysine residues of the antibody to unsubstituted amino groups. For example, as a linker, (antibody-NH)-CO-O-C 2 H 4 -SSL 2 -(uncharged hydrophilic polymer) {wherein L 2 is a binding or blood-stable is a linker}, when the disulfide bond in the linker is cleaved, the side chain amino group of the lysine residue of the antibody is restored by the following reaction. As a result, the modified antibody releases an unmodified antibody (intact antibody). In the following, antibodies of the invention in which the GLUT1 ligand is glucose (Gluc), the uncharged hydrophilic polymer is polyethylene glycol (PEG), and L 2 is —C 2 H 4 —O—CO— are tested in a reducing environment. shows the mechanism of dissociating from the linker and returning to the original antibody. This mechanism is similar when the GLUT1 ligand is a GLUT1 ligand other than glucose and the uncharged hydrophilic polymer is an uncharged hydrophilic polymer other than PEG.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
　開裂によって抗体のリジン残基の側鎖アミノ基からリンカー部分が解離し、当該アミノ基が非置換のアミノ基に戻るように構成されることによって、還元環境下において開裂可能なリンカーは、標的組織（例えば、脳実質または腫瘍組織）において、抗体を非置換の状態に戻すことができる。抗体の結合特性は、リジン残基の側鎖アミノ基を修飾すると低下する可能性があることが指摘されており、リジン残基の側鎖アミノ基が修飾されることによって抗体の結合特性が低下した場合であっても、当該抗体が標的組織（例えば、脳実質または腫瘍組織）の還元環境下に送達されると、抗体のリジン残基の側鎖アミノ基の修飾が外れ、非置換の状態に戻り、これにより抗体は、その結合特性を回復させ得る。 A linker cleavable in a reducing environment is configured such that cleavage dissociates the linker moiety from the side chain amino group of the lysine residue of the antibody and the amino group reverts to an unsubstituted amino group, thereby releasing the target tissue. (eg, in brain parenchyma or tumor tissue), the antibody can be returned to its undisplaced state. It has been pointed out that the binding properties of antibodies can be reduced by modifying the side chain amino groups of lysine residues, and modification of the side chain amino groups of lysine residues reduces the binding properties of antibodies. However, when the antibody is delivered to the target tissue (e.g., brain parenchyma or tumor tissue) in a reducing environment, the side chain amino group of the lysine residue of the antibody is unmodified and unsubstituted. to allow the antibody to regain its binding properties.
　ある態様では、修飾抗体のゼータ電位の絶対値が、未修飾抗体のゼータ電位の絶対値の１０％以下、５％以下、４％以下、３％以下、２％以下、または１％以下であり得る。ある態様では、修飾抗体のゼータ電位の絶対値が、未修飾抗体のゼータ電位の絶対値の３％～１％程度であり得る。修飾抗体は、非電荷親水性ポリマーブロックにより修飾されているために、そのゼータ電位の絶対値を低下させ得るのである。 In certain embodiments, the absolute zeta potential of the modified antibody is 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the absolute zeta potential of the unmodified antibody. obtain. In some embodiments, the absolute zeta potential of the modified antibody can be on the order of 3% to 1% of the absolute zeta potential of the unmodified antibody. A modified antibody can have a reduced absolute zeta potential because it is modified with an uncharged hydrophilic polymer block.
　ある態様では、修飾抗体は、未修飾抗体よりも平均流体力学径（体積平均）において大きく、例えば、１０％以上、２０％以上、３０％以上、４０％以上、５０％以上、６０％以上、または７０％以上大きい。ある態様では、修飾抗体は、未修飾抗体よりも平均流体力学径（体積平均）において４０％～８０％、または５０％～７０％大きい。 In some embodiments, the modified antibody is greater in mean hydrodynamic diameter (volume average) than the unmodified antibody, e.g. or greater than 70%. In some embodiments, the modified antibody has a mean hydrodynamic diameter (volume average) that is 40% to 80%, or 50% to 70% larger than the unmodified antibody.
　ある態様では、修飾抗体の抗原への結合性は、未未修飾抗体の抗原への結合性の２０％以下、１５％以下、１０％以下、９％以下、８％以下、７％以下、６％以下、５％以下、４％以下、３％以下、２％以下、または１％以下であり得る。 In some embodiments, the binding of the modified antibody to the antigen is 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6 % or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.
　本発明の抗体において、好ましい態様において、ＧＬＵＴ１リガンドは、グルコースであり得る。この態様において、グルコースは、例えば、その２位、３位または６位の炭素原子を介してＰＥＧと連結され得、ＧＬＵＴ１との好適に相互作用し得る。 In a preferred embodiment of the antibody of the present invention, the GLUT1 ligand can be glucose. In this aspect, glucose can be linked to PEG via, for example, its 2-, 3- or 6-position carbon atoms and can favorably interact with GLUT1.
　本発明の抗体において、ＰＥＧは、Mｗ（重量平均分子量）において、２，０００～１２，０００、例えば、５，０００であり得る。 In the antibody of the present invention, PEG can have a Mw (weight average molecular weight) of 2,000 to 12,000, for example, 5,000.
　本発明の抗体において、リジン残基の側鎖アミノ基（第１級アミン）のうちの１０～９０％、２０～８０％、３０～７０％、または４０～６０％が、ポリエチレングリコール（ＰＥＧ）によってリンカーを介して修飾され得る。本明細書では、リジン残基の側鎖アミノ基のうちのＰＥＧ修飾されたアミノ基の割合を「ＰＥＧ」の後に当該割合（％）を付して表すことがある。 In the antibodies of the present invention, 10-90%, 20-80%, 30-70%, or 40-60% of the side chain amino groups (primary amines) of lysine residues are polyethylene glycol (PEG) can be modified through a linker by In this specification, the proportion of PEG-modified amino groups among the side chain amino groups of lysine residues may be indicated by adding the proportion (%) after "PEG".
　本発明の抗体は、ＧＬＵＴ１リガンドで修飾されたＰＥＧによる修飾と、ＧＬＵＴ１リガンドで修飾されていないＰＥＧによる修飾の両方を受けていてもよい。本発明の抗体において、抗体を修飾するＰＥＧのうち、例えば、１０～１００％、３０～８０％、または４０～６０％のＰＥＧがＧＬＵＴ１で修飾され得る。本明細書では、ＰＥＧのうち、ＧＬＵＴ１リガンドで修飾されたＰＥＧの割合を「Ｇ」または「Ｇｌｕｃ」の後に当該割合（％）を付して表すことがある。 The antibody of the present invention may be modified with both GLUT1 ligand-modified PEG and non-GLUT1 ligand-modified PEG. In the antibodies of the present invention, of the PEGs that modify the antibodies, for example, 10-100%, 30-80%, or 40-60% of the PEGs can be modified with GLUT1. In this specification, the proportion of PEG modified with a GLUT1 ligand may be indicated by adding the proportion (%) after "G" or "Gluc".
　抗体の抗原が標的組織（例えば、脳実質）内の腫瘍の表面抗原または腫瘍組織内の細胞の表面抗原である場合、当該抗体は、抗体依存的細胞傷害活性（ＡＤＣＣ活性）および／または補体依存性細胞傷害活性（ＣＤＣ活性）を有する抗体とし得る。ＡＤＣＣ活性は、例えば、抗体のサブクラスをＩｇＧ１とすることにより増強され得る。抗体の抗原が標的組織（例えば、脳実質）内の腫瘍の表面抗原または腫瘍組織内の細胞の表面抗原である場合、当該抗体は、細胞傷害剤との抗体－薬物コンジュゲート（ＡＤＣ）の形態であってもよい。抗体は、例えば、非ＡＤＣの形態であってもよい。 When the antigen of the antibody is a surface antigen of a tumor within the target tissue (e.g., brain parenchyma) or a surface antigen of a cell within the tumor tissue, the antibody exhibits antibody-dependent cellular cytotoxicity (ADCC activity) and/or complement Antibodies with dependent cytotoxic activity (CDC activity) can be used. ADCC activity can be enhanced, for example, by subclassing the antibody to IgG1. When the antigen of the antibody is a surface antigen of a tumor within the target tissue (e.g., brain parenchyma) or a surface antigen of a cell within the tumor tissue, the antibody is in the form of an antibody-drug conjugate (ADC) with a cytotoxic agent. may be Antibodies can be, for example, in non-ADC forms.
　抗体は、ある態様では、抗体－薬物コンジュゲート（ＡＤＣ）の形態であり得る。この態様では、リンカーは抗体部分を修飾する。ある態様では、修飾は、十分な長さまたは大きさを有し、ＡＤＣのリンカー部分を包み込み、リンカーを分解から安定化させ得る、または、生体内の成分によるリンカーの分解を抑制し得る。修飾はまた、十分な長さまたは大きさを有し、ＡＤＣのリンカー部分および薬物部分を包みこみ、ＡＤＣの薬物部分が露出し、他成分と相互作用することによる副作用を低減し得る。修飾抗体部分を含むＡＤＣは、リンカー部分および薬物部分を１ＡＤＣあたりで、特に限定されないが例えば、１～１０個、２～８個、２～５個、２～４個、または３～４個程度含み得る。修飾抗体部分を含むＡＤＣは、１２ｎｍ以上、１３ｎｍ以上、１４ｎｍ以上、１５ｎｍ以上、１６ｎｍ以上、１７ｎｍ以上、約１８ｎｍ程度、および／または３０ｎｍ以下、２９ｎｍ以下、２８ｎｍ以下、２７ｎｍ以下、２６ｎｍ以下、または２５ｎｍ以下、例えば、１２～３０ｎｍ、または１５～２５ｎｍの平均流体力学半径（体積平均）を有し得る。ＡＤＣは、特に限定されないが例えば、ゲムツズマブ オゾガマイシン（マイロターグ）、イブリツモマブ チウキセタン（ゼヴァリン）、ブレンツキシマブ　ベドチン（アドセトリス）、トラスツズマブ　エムタンシン（カドサイラ）、イノツズマブ　オゾガマイシン（ベスポンサ）、モキセツモマブ　パスドトックス－ｔｄｆｋ（ＬＵＭＯＸＩＴＩ）、ポラツズマブ　ベドチン－Ｐｉｉｑ（Ｐｏｌｉｖｙ）、トラスツズマブ　デルクステカン（エンハーツ）、およびエンホルツマブ　ベドチン（ＰＡＤＣＥＶ）からなる群から選択される１以上を含んでいてもよい。 An antibody, in certain aspects, can be in the form of an antibody-drug conjugate (ADC). In this aspect, the linker modifies the antibody portion. In certain aspects, the modification is of sufficient length or size to envelop the linker portion of the ADC and may stabilize the linker from degradation or inhibit degradation of the linker by components in vivo. The modification may also be of sufficient length or size to envelop the linker portion and drug portion of the ADC, exposing the drug portion of the ADC and reducing side effects from interacting with other components. ADCs containing modified antibody moieties have linker moieties and drug moieties per 1 ADC, but are not particularly limited, for example, about 1 to 10, 2 to 8, 2 to 5, 2 to 4, or 3 to 4 can contain. ADCs comprising modified antibody moieties are 12 nm or greater, 13 nm or greater, 14 nm or greater, 15 nm or greater, 16 nm or greater, 17 nm or greater, about 18 nm or less, and/or 30 nm or less, 29 nm or less, 28 nm or less, 27 nm or less, 26 nm or less, or 25 nm. Below, for example, it may have an average hydrodynamic radius (volume average) of 12-30 nm, or 15-25 nm. ADCs include, but are not limited to, gemtuzumab ozogamicin (Milotarg), ibritumomab tiuxetan (Zevalin), brentuximab vedotin (ADCETRIS), trastuzumab emtansine (Kadcyra), inotuzumab ozogamicin (Vesponsa), moxetumomab pasudotox-tdfk (LUMOXITI), polatuzumab It may contain one or more selected from the group consisting of vedotin-Piiq (Polivy), trastuzumab deruxtecan (Enherts), and enfortumab vedotin (PADCEV).
　本発明の抗体は、イメージング剤と連結していてもよい。イメージング剤としては、例えば、生体適合性の蛍光色素（例えば、可視光領域の蛍光、または近赤外領域の蛍光を発する蛍光色素）および発光色素（例えば、ルシフェラーゼ）、並びに、放射性同位体、超音波プローブ、ＭＲＩ用造影剤およびＣＴ用造影剤などのイメージング剤が挙げられる。イメージング剤と抗体とのコンジュゲートは、当業者であれば適宜作製することができる。 The antibody of the present invention may be linked to an imaging agent. Imaging agents include, for example, biocompatible fluorescent dyes (e.g., fluorescent dyes that emit fluorescence in the visible light region or near-infrared region) and luminescent dyes (e.g., luciferase), and radioisotopes, ultra Imaging agents such as acoustic probes, MRI contrast agents and CT contrast agents are included. A conjugate of an imaging agent and an antibody can be appropriately prepared by those skilled in the art.
　本発明の抗体は、生理活性物質（例えば、酵素および核酸）と連結していてもよい。これにより、生理活性物質を標的組織（例えば、脳実質または腫瘍組織）に送達し得る。生理活性物質と抗体とのコンジュゲートは、当業者であれば適宜作製することができる。 The antibodies of the present invention may be linked to physiologically active substances (eg, enzymes and nucleic acids). This can deliver the bioactive agent to the target tissue (eg, brain parenchyma or tumor tissue). A conjugate of a physiologically active substance and an antibody can be appropriately prepared by those skilled in the art.
　本発明によれば、本発明の抗体を含んでなる、標的組織における疾患（例えば、脳疾患）を処置または予防するための医薬組成物が提供される。脳疾患としては、例えば、脳腫瘍が挙げられる。脳腫瘍としては、例えば、神経膠腫、神経膠芽腫、中枢神経系原発悪性リンパ腫、髄膜腫、下垂体腺腫、神経鞘腫、および頭蓋咽頭腫からなる群から選択される脳腫瘍が挙げられる。また、抗体としては、これら腫瘍に発現するＰＤ－Ｌ１に結合し、その活性を中和する抗体が挙げられる。抗体としては、これら腫瘍のリンパ球に発現するＰＤ－１に結合し、その活性を中和する抗体が挙げられる。抗体としては、これら腫瘍のリンパ球に発現するＣＴＬＡ４に結合し、その活性を中和する抗体が挙げられる。本明細書では、「病気の処置」は、病気の予防および病気の治療を意味する。病気の予防は、病気の発症を防止すること、発症を遅延させること、発症率を低下させること、を含む意味で用いられる。病気の治療は、病気の悪化の速度の低下、悪化の遅延、悪化の防止、病気の症状の軽減、病気の治癒、および病気の寛解を含み意味で用いられる。本発明によればまた、本発明の抗体を含んでなる、標的組織における疾患（例えば、がん）を処置または予防するための医薬組成物が提供される。がんとしては、肺がん（例えば、肺扁平上皮がん、肺腺がん、小細胞肺がん、非小細胞肺がん）、頭頸部がん、肝細胞がん、腎細胞がん、大腸がん、メラノーマ、膀胱がん、卵巣がん、胃がん、メルケル細胞がん、乳がん（例えば、トリプルネガティブ乳がん）、およびメルケル細胞がんが挙げられる。また、腫瘍としては、ホジキンリンパ腫が挙げられる。 According to the present invention, pharmaceutical compositions for treating or preventing diseases in target tissues (eg, brain diseases) are provided, comprising the antibodies of the present invention. Brain diseases include, for example, brain tumors. Brain tumors include, for example, brain tumors selected from the group consisting of glioma, glioblastoma, central nervous system primary malignant lymphoma, meningioma, pituitary adenoma, schwannoma, and craniopharyngioma. Antibodies also include antibodies that bind to PD-L1 expressed on these tumors and neutralize its activity. Antibodies include those that bind to PD-1 expressed on lymphocytes of these tumors and neutralize its activity. Antibodies include those that bind to CTLA4 expressed on lymphocytes of these tumors and neutralize its activity. As used herein, "treatment of disease" means prevention of disease and cure of disease. Disease prevention is used in the sense of preventing the onset of disease, delaying onset, and reducing the onset rate. Treatment of a disease is meant to include slowing the rate of exacerbation of the disease, delaying exacerbation, preventing exacerbation, alleviating the symptoms of the disease, curing the disease, and remission of the disease. The present invention also provides pharmaceutical compositions for treating or preventing disease (eg, cancer) in target tissues, comprising the antibodies of the present invention. Cancers include lung cancer (e.g. lung squamous cell carcinoma, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer), head and neck cancer, hepatocellular carcinoma, renal cell carcinoma, colon cancer, melanoma , bladder cancer, ovarian cancer, gastric cancer, Merkel cell carcinoma, breast cancer (eg, triple-negative breast cancer), and Merkel cell carcinoma. Tumors also include Hodgkin's lymphoma.
　本発明の抗体または医薬組成物は、投与計画に従って投与してもよい。ここで投与計画は、
　対象の血糖を低下させることと、その後、
　当該対象に対して、血糖値の上昇を誘発させない場合と比較してより多くの抗体が標的組織（例えば、脳実質）に移行するように、血糖値の上昇を誘発させること、および、本発明の抗体または医薬組成物を投与することを含み得る。ここで、対象は、血液脳関門を有する、または、機能破綻した血液脳関門を有する。機能破綻した血液脳関門は、抗体を含む高分子を脳実質に投下させる。したがって、機能破綻した血液脳関門を有する対象は、この投与計画無しで本発明の修飾抗体を投与し得る。 Antibodies or pharmaceutical compositions of the invention may be administered according to a dosing regimen. Here the dosing regimen is
lowering blood sugar in a subject, and thereafter,
Inducing an increase in blood glucose levels in the subject such that more antibodies are translocated to target tissues (e.g., brain parenchyma) compared to not inducing an increase in blood glucose levels, and the present invention administering an antibody or pharmaceutical composition of Here, the subject has a blood-brain barrier or has a dysfunctional blood-brain barrier. A dysfunctional blood-brain barrier allows macromolecules, including antibodies, to drop into the brain parenchyma. Thus, subjects with a compromised blood-brain barrier may be administered modified antibodies of the invention without this dosing regimen.
　本発明による投与計画では、該組成物は、該対象における血糖値の上昇の誘発と、同時に、連続してまたは逐次的に該対象に投与され得る。投与計画は、該対象への組成物の投与と該対象における血糖値の上昇の誘発との間にインターバルを有してもよいし、有さなくてもよい。該組成物が該対象における血糖値の上昇の誘発と同時に投与される場合には、該組成物は、血糖値の上昇の誘発を引き起こす薬剤と混合した形態で該対象に投与してもよいし、該対象における血糖値の上昇の誘発を引き起こす薬剤とは別の形態で投与してもよい。また、該組成物は、該対象における血糖値の上昇の誘発と、連続してまたは逐次的に該対象に投与される場合には、該組成物は該対象における血糖値の上昇の誘発より前に該対象に投与してもよいし、後に投与してもよいが、好ましくは、該組成物は該対象における血糖値の上昇の誘発より前に該対象に投与することができる。該対象への該組成物の投与よりも先に該対象において血糖値の上昇を誘発させる場合には、該対象において血糖値の上昇を誘発させてから、１時間以内、４５分以内、３０分以内、１５分以内または１０分以内に該対象に該組成物を投与することが好ましい。また、該対象への該組成物の投与よりも後に該対象において血糖値の上昇を誘発させる場合には、該対象に該組成物を投与してから、６時間以内、４時間以内、２時間以内、１時間以内、４５分以内、３０分以内、１５分以内または１０分以内に該対象において血糖値の上昇を誘発させることが好ましい。上記の投与計画のサイクルは、２回以上行なってもよい。グルコース投与とサンプル投与の前後関係は、血液脳関門を通過させるタイミングにより決定することができる。 In dosing regimens according to the present invention, the composition may be administered to the subject simultaneously, sequentially, or sequentially with the induction of elevated blood glucose levels in the subject. A dosing regimen may or may not have an interval between administering the composition to the subject and inducing an increase in blood glucose levels in the subject. When the composition is administered at the same time as inducing an increase in blood glucose level in the subject, the composition may be administered to the subject in a form mixed with an agent that induces an increase in blood glucose level. , may be administered in a form separate from the agent that causes the induction of elevated blood glucose levels in the subject. Also, the composition induces an increase in blood glucose levels in the subject and, if administered to the subject sequentially or sequentially, the composition prior to inducing an increase in blood glucose levels in the subject. It may be administered to the subject on or after, but preferably the composition is administered to the subject prior to inducing an increase in blood glucose levels in the subject. within 1 hour, within 45 minutes, or 30 minutes after inducing an increase in blood glucose level in the subject, when an increase in blood glucose level is induced in the subject prior to administration of the composition to the subject Preferably, the composition is administered to the subject within, within 15 minutes, or within 10 minutes. In addition, when an increase in blood glucose level is induced in the subject after administration of the composition to the subject, within 6 hours, within 4 hours, or 2 hours after administration of the composition to the subject Preferably, an increase in blood glucose level is induced in said subject within, within 1 hour, within 45 minutes, within 30 minutes, within 15 minutes or within 10 minutes. Two or more cycles of the above regimen may be performed. The context of glucose administration and sample administration can be determined by the timing of crossing the blood-brain barrier.
　本発明の別の態様では、ＧＬＵＴ１リガンドは、標的抗原に結合する分子に置き換えることができる。標的抗原は、例えば、細胞表面に発現する膜タンパク質であり得る。このようにすることにより、抗体を特定の細胞表面に集積し得る。抗原は、一例では、標的組織の細胞（例えば、脳の細胞）に発現する抗原であり得る。標的抗原に結合する分子としては、例えば、標的抗原に結合するタンパク質（例えば、抗体または環状タンパク質）、標的抗原に結合するアプタマー、および標的抗原に結合するレクチン（標的抗原が糖鎖を有する場合）があげられ、当業者であれば適宜選択するか、作成して本発明の標的抗原に結合する分子として用いることができる。 In another aspect of the invention, the GLUT1 ligand can be replaced with a molecule that binds to the target antigen. A target antigen can be, for example, a membrane protein expressed on the cell surface. By doing so, the antibody can be concentrated on the surface of specific cells. The antigen, in one example, can be an antigen expressed on cells of the target tissue (eg, cells of the brain). Molecules that bind to the target antigen include, for example, proteins that bind to the target antigen (e.g., antibodies or cyclic proteins), aptamers that bind to the target antigen, and lectins that bind to the target antigen (when the target antigen has sugar chains). Those skilled in the art can appropriately select or create and use them as molecules that bind to the target antigen of the present invention.
　本発明の医薬組成物は、本発明の抗体に加えて、医薬上許容可能な賦形剤をさらに含んでいてもよい。本発明の医薬組成物は、様々な形態、例えば、液剤（例えば注射剤）、分散剤、懸濁剤、錠剤、丸剤、粉末剤、坐剤などとすることができる。好ましい態様では、本発明の医薬組成物は、注射剤であり、非経口（例えば、静脈内、経皮、および腹腔内）で投与され得る。 The pharmaceutical composition of the present invention may further contain pharmaceutically acceptable excipients in addition to the antibody of the present invention. Pharmaceutical compositions of the present invention can be in various forms, such as liquids (eg, injections), dispersions, suspensions, tablets, pills, powders, suppositories, and the like. In a preferred embodiment, the pharmaceutical composition of the present invention is an injection and can be administered parenterally (eg, intravenously, transdermally, and intraperitoneally).
　本発明によれば、対象に本発明の抗体を投与することを含む、方法が提供される。本発明によれば、対象の標的組織（例えば、脳実質または腫瘍組織）に抗体を送達する方法であって、前記対象に本発明の抗体または当該抗体を含む医薬組成物を投与することを含む、方法が提供される。投与は、静脈内投与であり得る。これらの方法においては、本発明の抗体は、本発明による投与計画に従って投与され得る。本発明によれば、抗体を修飾する方法、およびこれにより本発明の修飾抗体を得る方法がそれぞれ提供される。 According to the present invention, a method is provided comprising administering the antibody of the present invention to a subject. According to the present invention, a method of delivering an antibody to a target tissue (e.g., brain parenchyma or tumor tissue) in a subject, comprising administering to said subject an antibody of the invention or a pharmaceutical composition comprising said antibody. , a method is provided. Administration can be intravenous. In these methods, antibodies of the invention can be administered according to dosing regimens according to the invention. According to the invention, methods for modifying antibodies and methods for obtaining modified antibodies of the invention are provided, respectively.
　本発明によれば、本発明の医薬組成物の製造における、本発明の抗体の使用が提供される。 According to the present invention, use of the antibody of the present invention in the manufacture of the pharmaceutical composition of the present invention is provided.
［材料と方法］
材料
　東京化成工業株式会社（東京、日本）から１，２：３，４－ジ－Ｏ－イソプロピリデン－α－Ｄ－グルコフラノシド（ＤＩＧ）を購入した。α－メトキシ－ω－アミノポリエチレングリコール（ＭｅＯ－ＰＥＧ－ＮＨ2）（ＰＥＧのＭｗは５，５００）を、日油株式会社（東京、日本）から購入した。２－［（２－［（４－ニトロフェノキシ）カルボニル］オキシエチル）ジスルファニル］エチル４－ニトロフェニルカーボネート（ＮＰＣ－（ＣＨ2）2－Ｓ－Ｓ－（ＣＨ2）2－ＮＰＣ（ＮＰＣ：　ｐ－ニトロフェニルカーボネート）は、Ｅｎａｍｉｎｅ　Ｃｏ．，　Ｌｔｄ（東京都）より入手した。Ａｌｅｘａ　ＦｌｕｏｒTM　６４７　ＮＨＳエステル（スクシンイミジルエステル）は、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ　（ＭＡ，　ＵＳＡ）から購入した。フルオレスカミン、Ｌ－グルタチオン還元物はＳｉｇｍａ－Ａｌｄｒｉｃｈ　Ｃｏ．（Ｓｔ．　Ｌｏｕｉｓ，　ＭＯ）から入手した。本研究に使用した有機溶媒は、ニッコー・ハンセン株式会社（大阪、日本）から購入したものである。有機溶媒は、中性アルミナの２本のカラムを通過させることで精製した。ＣｅｌｌＭａｓｋTM　Ｇｒｅｅｎ　Ｐｌａｓｍａ　Ｍｅｍｂｒａｎｅ　Ｓｔａｉｎ　（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ，　カタログ番号　Ｃ３７６０８）とＨｏｅｃｈｓｔ　３３３４２　（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ，　カタログ番号　６２２４９）を使用して、細胞サンプルと組織スライスの細胞膜と核を染色した。 [Materials and methods]
Materials 1,2:3,4-di-O-isopropylidene-α-D-glucofuranoside (DIG) was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). α-Methoxy-ω-amino polyethylene glycol (MeO-PEG-NH 2 ) (PEG Mw is 5,500) was purchased from NOF Corporation (Tokyo, Japan). 2-[(2-[(4-nitrophenoxy)carbonyl]oxyethyl)disulfanyl]ethyl 4-nitrophenyl carbonate (NPC-(CH 2 ) 2 -SS-(CH 2 ) 2 -NPC(NPC: p -nitrophenyl carbonate) was obtained from Enamine Co., Ltd., Tokyo, Japan Alexa Fluor ™ 647 NHS ester (succinimidyl ester) was purchased from Thermo Fisher Scientific (MA, USA) Fluorescamine, L-glutathione reduced product was obtained from Sigma-Aldrich Co. (St. Louis, Mo.) Organic solvents used in this study were purchased from Nikko Hansen Co., Ltd. (Osaka, Japan). was purified by passage through two columns of neutral alumina, using CellMask ™ Green Plasma Membrane Stain (Thermo Fisher Scientific, Catalog No. C37608) and Hoechst 33342 (Thermo Fisher Scientific, Catalog No. 62249) to Cell samples and tissue slices were stained for cell membranes and nuclei.
細胞株
　ルシフェラーゼでタグ付けされたマウス神経膠芽腫細胞株－ＧＬ２６１およびルシフェラーゼでタグ付けされたＣＴ２Ａは、ＪＣＲＢ細胞バンク（大阪、日本）から入手した。ＧＬ２６１およびＣＴ２Ａ細胞は、１０％ウシ胎児血清（Ｉｎｖｉｔｒｏｇｅｎ）、１００Ｕ　ｍｌ-1ペニシリン（Ｉｎｖｉｔｒｏｇｅｎ）および１００Ｕ　ｍｌ-1ストレプトマイシン（Ｉｎｖｉｔｒｏｇｅｎ）を補充したＲＰＭＩ１６４０培地（Ｇｉｂｃｏ；Ｉｎｖｉｔｒｏｇｅｎ）中で維持した。細胞は、５％ＣＯ2、相対湿度９０％の雰囲気下、３７℃のインキュベーター（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｃｉｆｅｒ　Ｓｃｉｅｎｃｉｆｉｃ）で培養した。 Cell lines luciferase-tagged mouse glioblastoma cell line-GL261 and luciferase-tagged CT2A were obtained from the JCRB cell bank (Osaka, Japan). GL261 and CT2A cells were maintained in RPMI 1640 medium (Gibco; Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 U ml −1 penicillin (Invitrogen) and 100 U ml −1 streptomycin (Invitrogen). Cells were cultured in a 37° C. incubator (Thermo Fisher Scientific) in an atmosphere of 5% CO 2 and 90% relative humidity.
抗体
　臨床承認されたヒト抗ＰＤ－Ｌ１抗体（ａＰＤ－Ｌ１）であるアベルマブ（商品名ＢＡＶＥＮＣＩＯ）は、Ｍｅｒｃｋ　＆　Ｃｏ．から商業的に入手した。免疫染色に使用した抗体は、ＣＤ３（ＢｉｏＬｅｇｅｎｄ；カタログ番号１００２０５、クローン：１７Ａ２）、ＣＤ４（ＢｉｏＬｅｇｅｎｄ；カタログ番号１００４３４、クローン：ＧＫ１．５）、ＣＤ４５（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ；カタログ番号５５３０８０、クローン：３０－Ｆ１１）、ＣＤ８ａ（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ；カタログ番号５５３０３５、クローン：５３－６．７）、ＦＯＸＰ３（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ；カタログ番号５６０４０８、クローン：ＭＦ２３）、ＣＤ４４（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ；カタログ番号５５３１３３、クローン：ＩＭ７）およびＣＤ６２Ｌ（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ；カタログ番号５５３１５２、クローン：ＭＥＬ－１４）。多色フローサイトメトリーは、適切な補正を行って使用した。すべての抗体は、製造業者の指示書に従って使用した。抗体にコンジュゲートされた蛍光色素は、同じ蛍光色素のチャンネルに正確に一致した。染色後、細胞をＦｌｏｗＪｏソフトウェアパッケージを用いて分析した。組換えマウスＰＤＬ１タンパク質は、Ａｂｃａｍ（カタログ番号ａｂ１３００３９）から購入した。免疫染色およびＥＬＩＳＡに使用した二次抗体は、ヤギ抗ヒトＩｇＧ　Ｈ＆Ｌ（Ａｂｃａｍ、カタログ番号ａｂ９７１７５）およびヤギ抗ヒトＩｇＧ（Ｈ＋Ｌ）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｃｉｆｅｒ　Ｓｃｉｅｎｃｉｆｉｃ、カタログ番号ａ－１１０１３）であった。
マウス
　Ｃ５７ＢＬ／６Ｊマウス（雌；６週齢または７週齢）をチャールズリバー社（東京都）から購入した。東京、日本）から購入した。すべての動物実験は、実施施設であるナノ医療イノベーションセンターの倫理指針に基づいて実施した。 Avelumab (trade name BAVENCIO), a clinically approved human anti-PD-L1 antibody (aPD-L1), is available from Merck & Co. was commercially obtained from Antibodies used for immunostaining are CD3 (BioLegend; catalog number 100205, clone: 17A2), CD4 (BioLegend; catalog number 100434, clone: GK1.5), CD45 (BD Biosciences; catalog number 553080, clone: 30-F11 ), CD8a (BD Biosciences; Catalog No. 553035, Clone: 53-6.7), FOXP3 (BD Biosciences; Catalog No. 560408, Clone: MF23), CD44 (BD Biosciences; Catalog No. 553133, Clone: IM7) and CD62L ( BD Biosciences; catalog number 553152, clone: MEL-14). Multicolor flow cytometry was used with appropriate corrections. All antibodies were used according to the manufacturer's instructions. The fluorochromes conjugated to the antibodies were matched exactly to the channels of the same fluorochrome. After staining, cells were analyzed using the FlowJo software package. Recombinant mouse PDL1 protein was purchased from Abcam (catalog number abl30039). The secondary antibodies used for immunostaining and ELISA were goat anti-human IgG H&L (Abcam, catalog number ab97175) and goat anti-human IgG (H+L) (Thermo Fisher Scientific, catalog number a-11013).
Mouse C57BL/6J mice (female; 6-week old or 7-week old) were purchased from Charles River (Tokyo). Tokyo, Japan). All animal experiments were conducted in accordance with the ethical guidelines of the facility, the Innovation Center of NanoMedicine.
ＭｅＯ－ＰＥＧ－ＮＨ－Ｃ（Ｏ）－Ｏ－（ＣＨ 2 ） 2 －Ｓ－Ｓ－（ＣＨ 2 ） 2 －ＮＰＣとＤＩＧ－ＰＥＧ－ＮＨ－Ｃ（Ｏ）－Ｏ－（ＣＨ 2 ） 2 －Ｓ－Ｓ－（ＣＨ 2 ） 2 －ＮＰＣの合成
　まず、ＤＩＧ－ＰＥＧ－ＮＨ2は、先行論文に従って合成した。具体的には、ジクロロメタン（ＤＣＭ）に溶解したＭｅＯ－ＰＥＧ－ＮＨ2（１．０　ｇ，　０．１６７　ｍｍｏｌ）またはＤＩＧ－ＰＥＧ－ＮＨ2（１．０　ｇ，　０．１６７　ｍｍｏｌ）をＮＰＣ－（ＣＨ2）2－Ｓ－Ｓ－（ＣＨ2）2－ＮＰＣ（３２４　ｍｇ，　０．６６７　ｍｍｏｌ）に氷浴下で１０分間添加した。冷たい混合物にピリジン（５４μＬ，　０．６６７　ｍｍｏｌ）を激しく撹拌しながら滴下添加した。その後、反応混合物を室温で一晩撹拌し、続いて、ＭＷＣＯ　３．５ｋＤａの膜を用いてＤＭＳＯに対して透析した。最後に、ＭｅＯ－ＰＥＧ－ＮＨ－Ｃ（Ｏ）－Ｏ－（ＣＨ2）2－Ｓ－Ｓ－（ＣＨ2）2－ＮＰＣおよびＤＩＧ－ＰＥＧ－ＮＨ－Ｃ（Ｏ）－Ｏ－（ＣＨ2）2－Ｓ－Ｓ－（ＣＨ2）2－ＮＰＣを、さらなる用途のために凍結乾燥した。
　ＤＩＧ－ＰＥＧ－ＮＨ－Ｃ（Ｏ）－Ｏ－（ＣＨ2）2－Ｓ－Ｓ－（ＣＨ2）2－ＮＰＣ（１０．０　ｍｇ）にＴＦＡ（０．５　ｍＬ，　ＴＦＡ／水　＝　４／１，　ｖ／ｖ）を加え、室温で３０分間激しく攪拌した。次に、この溶液に蒸留水１０．０ｍＬを氷浴中でゆっくりと加えた。次に、得られた溶液を水、１０　ｍＭ　ＨＣｌ、および蒸留水に対して順次透析した。最後に、精製溶液を凍結乾燥し、得られたポリマーの1Ｈ－ＮＭＲをＤ2Ｏ中で２５℃で測定した。得られたポリマーは、ＤＩＧがグルコースに脱保護されたＧｌｕｃ－ＰＥＧ－ＮＨ－Ｃ（Ｏ）－Ｏ－（ＣＨ2）2－Ｓ－Ｓ－（ＣＨ2）2－ＮＰＣであった。
　コントロールポリマーとして、非開裂性の架橋を形成するＮＰＣ－ＣＨ2）2－ＣＨ2－ＣＨ2－ＣＨ2－（ＣＨ2）2－ＮＰＣを用い、上記と同様の合成工程に従って、ＭｅＯ－ＰＥＧ－ＮＨ－Ｃ（Ｏ）－Ｏ－（ＣＨ2）2－ＣＨ2－ＣＨ2－ＮＰＣおよびＤＩＧ－ＰＥＧ－ＮＨ－Ｃ（Ｏ）－Ｏ－（ＣＨ2）2－ＣＨ2－ＣＨ2－ＣＨ2－（ＣＨ2）2－ＮＰＣを調製した。 MeO-PEG-NH-C(O)-O-(CH2 ) 2 - SS- (CH2 ) 2 - NPC and DIG-PEG-NH-C(O)-O- ( CH2 ) 2- Synthesis of SS- (CH 2 ) 2 -NPC First, DIG-PEG-NH 2 was synthesized according to the previous paper. Specifically, MeO-PEG-NH 2 (1.0 g, 0.167 mmol) or DIG-PEG-NH 2 (1.0 g, 0.167 mmol) dissolved in dichloromethane (DCM) was added to NPC- (CH 2 ) 2 --S—S—(CH 2 ) 2 --NPC (324 mg, 0.667 mmol) was added under an ice bath for 10 minutes. Pyridine (54 μL, 0.667 mmol) was added dropwise to the cold mixture with vigorous stirring. The reaction mixture was then stirred overnight at room temperature and subsequently dialyzed against DMSO using a MWCO 3.5 kDa membrane. Finally, MeO-PEG-NH-C(O)-O-(CH 2 ) 2 -SS-(CH 2 ) 2 -NPC and DIG-PEG-NH-C(O)-O-(CH 2 ) 2 -SS-(CH 2 ) 2 -NPC was lyophilized for further use.
DIG-PEG-NH-C(O)-O-(CH 2 ) 2 -S-S-(CH 2 ) 2 -NPC (10.0 mg) was added with TFA (0.5 mL, TFA/water = 4/ 1, v/v) was added and vigorously stirred at room temperature for 30 minutes. Next, 10.0 mL of distilled water was slowly added to this solution in an ice bath. The resulting solution was then dialyzed sequentially against water, 10 mM HCl, and distilled water. Finally, the purified solution was lyophilized and the 1 H-NMR of the resulting polymer was measured in D 2 O at 25°C. The resulting polymer was Gluc-PEG-NH-C(O)-O-(CH 2 ) 2 -SS-(CH 2 ) 2 -NPC with DIG deprotected to glucose.
Using NPC-CH 2 ) 2 -CH 2 -CH 2 -CH 2 -(CH 2 ) 2 -NPC, which forms non-cleavable crosslinks, as a control polymer, MeO-PEG- NH-C(O)-O-( CH2 ) 2 - CH2 - CH2 -NPC and DIG-PEG-NH-C(O)-O-( CH2 ) 2 - CH2 - CH2 - CH2 —(CH 2 ) 2 —NPC was prepared.
様々なグルコース密度のＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１の調製
　リン酸緩衝液（ｐＨ８．４、２０ｍＭ）に１００％：０％、５０％：５０％、２５％：７５％、０％：１００％を含む様々な供給比で溶解したＧｌｕｃ－ＰＥＧ－（ＣＨ2）2－Ｓ－Ｓ－（ＣＨ2）2－ＮＰＣ（１０．０　ｍｇ　ｍＬ-1）とＭｅＯ－ＰＥＧ－（ＣＨ2）2－Ｓ－Ｓ－（ＣＨ2）2－ＮＰＣ（１０．０　ｍｇ　ｍＬ-1）を含む１．０　ｍＬ溶液をａＰＤ－Ｌ１（１．０　ｍｇ）と混合し、室温で一晩かけて共有結合を行った。次に、この溶液をＶｉｖａｓｐｉｎ　６（３回、カットオフＭＷ：３０　ｋＤａ、２０　ｍＭ　ｐＨ　７．４リン酸緩衝液）で精製し、未反応ポリマーを除去して、ａＰＤ－Ｌ１が、ジスルフィド結合を介してグルコースと連結した抗体複合体Ｇｌｕｃ－ＰＥＧ－ＮＨ－Ｃ（Ｏ）－Ｏ－（ＣＨ2）2－Ｓ－Ｓ－（ＣＨ２）２－Ｏ－Ｃ（Ｏ）－ＮＨ－ａＰＤ－Ｌ１（以下、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１と表すことがある。）を得た。このようにして、０、２５、５０および１００％の様々なグルコース密度（グルコースの表面密度）を有するＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１を調製し、さらなる利用のために保存した。抗体のＰＥＧ－ＮＨ－Ｃ（Ｏ）－Ｏ－（ＣＨ2）2－Ｓ－Ｓ－（ＣＨ2）2－ＮＰＣ（以下、ＰＥＧ－Ｓ－ＮＰＣと表すことがある。）による修飾は、抗体がその結合親和性を有意に低下させる、または、喪失する強度で行うことができる。 Preparation of Gluc-S-aPD-L1 with various glucose densities 100%:0%, 50%:50%, 25%:75%, 0%:100% in phosphate buffer (pH 8.4, 20 mM) Gluc-PEG-(CH 2 ) 2 -SS-(CH 2 ) 2 -NPC (10.0 mg mL −1 ) and MeO-PEG-(CH 2 ) 2 -S dissolved at various feed ratios containing A 1.0 mL solution containing -S-(CH 2 ) 2 -NPC (10.0 mg mL -1 ) was mixed with aPD-L1 (1.0 mg) and covalently coupled overnight at room temperature. rice field. This solution was then purified on Vivaspin 6 (three times, cut-off MW: 30 kDa, 20 mM pH 7.4 phosphate buffer) to remove unreacted polymer and to allow aPD-L1 to cross disulfide bonds. Antibody Conjugate Gluc-PEG-NH-C(O)-O-(CH 2 ) 2 -S-S-(CH 2 ) 2 -O-C(O)-NH-aPD-L1 Linked to Glucose Via (hereinafter sometimes referred to as Gluc-S-aPD-L1). Gluc-S-aPD-L1 with various glucose densities (surface density of glucose) of 0, 25, 50 and 100% were thus prepared and stored for further use. Modification of an antibody with PEG-NH-C(O)-O-(CH 2 ) 2 -S-S-(CH 2 ) 2 -NPC (hereinafter sometimes referred to as PEG-S-NPC) is can be performed at an intensity that significantly reduces or loses its binding affinity.
Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１の特性評価
　ａＰＤ－Ｌ１とＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１のサイズ分布とゼータ電位は、ゼータサイザー　Ｎａｎｏ　ＺＳ９０（Ｍａｌｖｅｒｎ　Ｉｎｓｔｒｕｍｅｎｔｓ　Ｌｔｄ．、Ｗｏｒｃｅｓｔｅｒｓｈｉｒｅ、英国）を使用してｐＨ７．４で１０ｍＭリン酸緩衝液で２５℃でのＤＬＳ測定によって評価した。ＰＥＧ修飾率を決定するために、アセトンに溶解したフルオレスカミン（３０．０μＬ、３．０ｍｇ　ｍＬ-1）をＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１（９０μＬ、１．０～１０．０μｇ　ｍＬ-1）と混合し、６６６ｎｍでの蛍光強度を検出して、ヒト血清アルブミンをコントロール（１分子あたり６０アミノ基）として使用してａＰＤ－Ｌ１の表面上の残りのアミン基を計算した。ａＰＤ－Ｌ１およびＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１をＧＳＨ（１．０　ｍＭ）の存在下または非存在下で様々な時間インキュベートした場合のゲル浸透クロマトグラフィー（ＧＰＣ）スペクトルを、サイズ排除クロマトグラフィー（Ｓｕｐｅｒｏｓｅ　６　Ｉｎｃｒｅａｓｅ　１０／３００カラム、ＧＥ）を用いて行った。分子量変化を評価するために、４０分以内に２８０ｎｍでの吸光度を記録した。ａＰＤ－Ｌ１およびＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１をＧＳＨ（１．０　ｍＭ）の存在下または非存在下でインキュベートした場合の円二色性スペクトルを、ＣＤスペクトロメーター（ＪＡＳＣＯ　Ｊ－８１５）を用いて室温で測定した。 Characterization of Gluc-S-aPD-L1 Size distribution and zeta potential of aPD-L1 and Gluc-S-aPD-L1 were determined at pH 7.4 using a Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, UK). was evaluated by DLS measurements at 25 °C in 10 mM phosphate buffer. To determine the percent PEG modification, fluorescamine (30.0 μL, 3.0 mg mL −1 ) dissolved in acetone was added to Gluc-S-aPD-L1 (90 μL, 1.0-10.0 μg mL −1 ). and detecting fluorescence intensity at 666 nm to calculate the remaining amine groups on the surface of aPD-L1 using human serum albumin as a control (60 amino groups per molecule). Gel permeation chromatography (GPC) spectra of aPD-L1 and Gluc-S-aPD-L1 incubated in the presence or absence of GSH (1.0 mM) for various times were analyzed by size exclusion chromatography (Superose 6 Increase 10/300 columns, GE). Absorbance at 280 nm was recorded within 40 minutes to assess molecular weight changes. Circular dichroism spectra of aPD-L1 and Gluc-S-aPD-L1 incubated in the presence or absence of GSH (1.0 mM) were analyzed using a CD spectrometer (JASCO J-815). Measured at room temperature.
Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１の還元環境応答型の活性の回復（再活性化）
　ａＰＤ－Ｌ１およびＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１とＰＤ－Ｌ１タンパク質との結合親和性を評価するために、組換えマウスＰＤ－Ｌ１タンパク質を９６ウェルプレートの底面にコーティングし、被検抗体（ａＰＤ－Ｌ１またはＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１）およびこれに対するＨＲＰ標識二次抗体を反応させ４５０ｎｍでの吸光度を記録することにより、抗原に対する抗体の特異的な結合量を決定するＥＬＩＳＡ法を開発した。また、ａＰＤ－Ｌ１およびＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１をＧＳＨ（１．０　ｍＭ）の存在下または非存在下で様々な時間プレインキュベートしてジスルフィド結合を開裂させた後、ＰＤ－Ｌ１タンパク質と反応させることによって、上記系を用いて活性の回復率を算出した。 Restoration of reducing environment-responsive activity of Gluc-S-aPD-L1 (reactivation)
To assess the binding affinity of aPD-L1 and Gluc-S-aPD-L1 to the PD-L1 protein, recombinant mouse PD-L1 protein was coated on the bottom of a 96-well plate and the antibody to be tested (aPD- L1 or Gluc-S-aPD-L1) and an HRP-labeled secondary antibody against it were reacted and the absorbance at 450 nm was recorded to develop an ELISA method to determine the specific binding amount of the antibody to the antigen. Also, aPD-L1 and Gluc-S-aPD-L1 were preincubated in the presence or absence of GSH (1.0 mM) for various times to cleave disulfide bonds prior to reaction with PD-L1 protein. The rate of recovery of activity was calculated using the above system.
　ａＰＤ－Ｌ１およびＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１と腫瘍細胞表面に発現したＰＤ－Ｌ１タンパク質との特異的な結合を観察するために、ＣｅｌｌＭａｓｋTM　Ｇｒｅｅｎ　Ｐｌａｓｍａ　Ｍｅｍｂｒａｎｅ　ＳｔａｉｎでＧＬ２６１腫瘍細胞の細胞膜を染色し、ＧＳＨ（１．０　ｍＭ）の存在下または非存在下で処理したａＰＤ－Ｌ１およびＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１をＧＬ２６１細胞とさらに１時間インキュベートし、最後に共焦点レーザー走査顕微鏡（ＬＳＭ　８８０、Ｚｅｉｓｓ）を用いて抗体の結合親和性を評価した。 In order to observe the specific binding of aPD-L1 and Gluc-S-aPD-L1 to PD-L1 protein expressed on the surface of tumor cells, the cell membrane of GL261 tumor cells was stained with CellMask ™ Green Plasma Membrane Stain, aPD-L1 and Gluc-S-aPD-L1 treated with or without GSH (1.0 mM) were incubated with GL261 cells for an additional hour and finally analyzed by confocal laser scanning microscopy (LSM 880, Zeiss ) was used to assess the binding affinity of the antibodies.
　ａＰＤ－Ｌ１およびＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１とＰＤ－Ｌ１タンパク質との結合量を定量的に測定するために、ＧＬ２６１腫瘍細胞を、ＧＳＨ（１．０　ｍＭ）の存在下または非存在下で処理したａＰＤ－Ｌ１およびＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１と一緒に１時間インキュベートし、３回洗浄した。次に、ＡＰＣ標識抗マウスＰＤ－Ｌ１抗体をＧＬ２６１細胞と１時間さらにインキュベートし、その後、ＦｌｏｗＪｏソフトウェアパッケージを用いた分析を行い、残りのＰＤ－Ｌ１タンパク質を測定した。 To quantitatively measure the amount of aPD-L1 and Gluc-S-aPD-L1 bound to PD-L1 protein, GL261 tumor cells were treated with or without GSH (1.0 mM). aPD-L1 and Gluc-S-aPD-L1 for 1 hour and washed 3 times. APC-labeled anti-mouse PD-L1 antibody was then further incubated with GL261 cells for 1 hour, followed by analysis using the FlowJo software package to measure residual PD-L1 protein.
Ｉｎ　ｖｉｖｏでの薬物動態と生体内分布
　すべての動物実験は、ナノ医療イノベーションセンターの倫理指針に基づいて実施した。なお、本実施例では、いずれの実験においても、血糖操作は行っていない。薬物動態を調べるために、様々なグルコース密度を有するａＰＤ－Ｌ１及びＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１を健康なＢＡＬＢ／ｃマウスに尾静脈を介して１．０　ｍｇ　ｋｇ-1の用量で投与た。投与後１時間、４時間、１２時間、および２４時間の時点で血液サンプルを採取した。血液サンプルを抽出し、ＧＳＨ（１０　ｍＭ）の存在下または非存在下で処理した。その後、上記で設計したＥＬＩＳＡプロトコルを用いて、ａＰＤ－Ｌ１の濃度を求めた。
　生理分布を実証するために、Ａｌｅｘａ６４７標識ａＰＤ－Ｌ１および様々なグルコース密度を有するＡｌｅｘａ　６４７標識Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１（１．０　ｍｇ　ｋｇ-1）を、同所性ＧＬ２６１腫瘍を有するＣ５７ＢＬ／６Ｊマウスに静脈内投与し、心臓、肝臓、脾臓、肺、腎臓、脳および腫瘍の抽出のために１時間、４時間、１２時間および２４時間後に犠牲にした。次に、各種組織をＤ－ＰＢＳ（－）で洗浄し、過剰な洗浄液を除去した後、重量を測定し、６００μＬの細胞溶解緩衝液でホモジナイズした。最後に、ａＰＤ－Ｌ１およびＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１の生物分布を、Ｉｎｆｉｎｉｔｅ　Ｍ１０００　ＰＲＯ分光光度計（Ｔｅｃａｎ　Ｇｒｏｕｐ　Ｌｔｄ．、Ｍａｎｎｅｄｏｒｆ、スイス）を用いた蛍光測定により定量した。 In Vivo Pharmacokinetics and Biodistribution All animal experiments were conducted in accordance with the ethical guidelines of the Innovation Center of Nanomedicine. In this example, blood sugar manipulation was not performed in any experiment. To study pharmacokinetics, aPD-L1 and Gluc-S-aPD-L1 with different glucose densities were administered to healthy BALB/c mice via the tail vein at a dose of 1.0 mg kg −1 . Blood samples were taken at 1 hour, 4 hours, 12 hours and 24 hours after dosing. Blood samples were extracted and treated with or without GSH (10 mM). The concentration of aPD-L1 was then determined using the ELISA protocol designed above.
To demonstrate physiological distribution, Alexa 647-labeled aPD-L1 and Alexa 647-labeled Gluc-S-aPD-L1 (1.0 mg kg −1 ) with varying glucose densities were administered to C57BL/L1 with orthotopic GL261 tumors. 6J mice were dosed intravenously and sacrificed 1, 4, 12 and 24 hours later for heart, liver, spleen, lung, kidney, brain and tumor extraction. Next, each tissue was washed with D-PBS(-), excess washing solution was removed, weighed, and homogenized with 600 μL of cell lysis buffer. Finally, the biodistribution of aPD-L1 and Gluc-S-aPD-L1 was quantified by fluorescence measurements using an Infinite M1000 PRO spectrophotometer (Tecan Group Ltd., Mannedorf, Switzerland).
還元環境応答性のＧｌｕｃ－ＰＥＧ鎖切断のｉｎ　ｖｉｖｏ観察
　Ａｌｅｘａ　６４７標識ａＰＤ－Ｌ１、Ａｌｅｘａ　６４７標識Ｇｌｕｃ２５－Ｃ－ａＰＤ－Ｌ１およびＡｌｅｘａ　６４７標識Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１を、同所性ＧＬ２６１腫瘍を有するマウスに静脈内投与し、これを２４時間後に犠牲にして腫瘍組織を抽出した。その後、腫瘍を１０．０μｍ切片に切断し、これを冷アセトンで固定し、ＰＢＳで洗浄し、５％ＢＳＡで室温で１時間ブロッキングした。その後、切片をＡｌｅｘａ　４８８標識抗ヒトＩｇＧ（Ｈ＋Ｌ）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ、カタログ番号Ａ－１１０１３）で４℃で一晩インキュベートし、その後、ＣＬＳＭ観察を行った。 In vivo observation of reducing environment-responsive Gluc-PEG chain scission mice were intravenously administered and sacrificed 24 hours later to extract tumor tissue. Tumors were then cut into 10.0 μm sections, which were fixed with cold acetone, washed with PBS, and blocked with 5% BSA for 1 hour at room temperature. Sections were then incubated with Alexa 488-labeled anti-human IgG (H+L) (Thermo Fisher Scientific, Catalog No. A-11013) overnight at 4° C., followed by CLSM observation.
同所性脳腫瘍のｉｎ　ｖｉｖｏ治療効果
　ルシフェラーゼ（ｌｕｃ）を発現するＧＬ２６１－ｌｕｃまたはＣＴ２Ａ－ｌｕｃ細胞（２μＬの注入量で１．０×１０5細胞）を、Ｃ５７ＢＬ／６Ｊマウスの脳内に、ブレグマの前方１．０ｍｍ、右側２．０ｍｍ、深さ３．０ｍｍの位置で頭蓋内に接種した。マウスに細胞を注入した日を０日目と定義した。腫瘍結節ができたら、マウス（ｎ＝５）を無作為化し、各種製剤（ａＰＤ－Ｌ１については１．５　ｍｇ　ｋｇ-1）の単回注射を尾静脈で投与した。ルシフェリン１５０ｍｇ　ｋｇ-1の注入後、ＧＬ２６１－ｌｕｃまたはＣＴ２Ａ－ｌｕｃ腫瘍からの生物発光シグナルをｉｎ　ｖｉｖｏイメージングシステム（ＩＶＩＳ）で観察した。マウスの生存期間を追跡し、その延長の有意性をログランク検定により決定した。 In vivo therapeutic effect of orthotopic brain tumors GL261-luc or CT2A-luc cells expressing luciferase (luc) (1.0 × 10 5 cells in a 2 μL injection volume) were injected into the brains of C57BL/6J mice at bregma. It was inoculated intracranially at 1.0 mm anterior, 2.0 mm right, and 3.0 mm deep. Day 0 was defined as the day mice were injected with cells. Once tumor nodules were established, mice (n=5) were randomized and given a single injection of various formulations (1.5 mg kg −1 for aPD-L1) via the tail vein. Bioluminescence signals from GL261-luc or CT2A-luc tumors were observed with an in vivo imaging system (IVIS) after injection of 150 mg kg −1 of luciferin. Mouse survival time was followed and the significance of the prolongation was determined by log-rank test.
　記憶免疫の成立の確認のための腫瘍の再チャレンジ試験のために、Ｃ５７ＢＬ／６Ｊマウスを、１．０×１０5個のＧＬ２６１細胞を最初に注入と、続くＧｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１での処理から９０日後に注入した。その後、生物発光シグナルをモニターし、生存時間を記録した。 For tumor rechallenge studies to confirm the establishment of memory immunity, C57BL/6J mice were first injected with 1.0×10 5 GL261 cells followed by Gluc25-S-aPD-L1. Injections were made 90 days after treatment. Bioluminescence signals were then monitored and survival time recorded.
Ｔ細胞分析およびＩＮＦγ検出
　ＩＦＮγの血漿レベルは、ＩＦＮγ　ＥＬＩＳＡキット（ＢｉｏＬｅｇｅｎｄ；カタログ番号４３０８０４）を用いて測定した。末梢血を、ａＰＤ－Ｌ１、Ｇｌｕｃ０－Ｓ－ａＰＤ－Ｌ１およびＧｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１の注射後３日目に採取し、５００ｇで１０分間遠心分離した。上清を分注し、分析まで－８０℃で保存した。サンプルは製造元の指示書に従いＥＬＩＳＡアッセイバッファーで希釈し、ＥＬＩＳＡキットを用いて分析した。 T-cell analysis and INFγ detection Plasma levels of IFNγ were measured using the IFNγ ELISA kit (BioLegend; Catalog No. 430804). Peripheral blood was collected 3 days after injection of aPD-L1, Gluc0-S-aPD-L1 and Gluc25-S-aPD-L1 and centrifuged at 500 g for 10 minutes. Supernatants were aliquoted and stored at -80°C until analysis. Samples were diluted in ELISA assay buffer according to the manufacturer's instructions and analyzed using an ELISA kit.
　Ｔ細胞分析のために、腫瘍組織をａＰＤ－Ｌ１、Ｇｌｕｃ０－Ｓ－ａＰＤ－Ｌ１およびＧｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１の注射後３日目に切除し、ＢＤ　ＨｏｒｉｚｏｎTM　Ｄｒｉ　Ｔｉｓｓｕｅ　＆　Ｔｕｍｏｕｒ　Ｄｉｓｓｏｃｉａｔｉｏｎ　Ｒｅａｇｅｎｔで解離させた。次いで、腫瘍細胞を抗ＣＤ３抗体、抗ＣＤ８抗体、抗ＣＤ４抗体、抗ＣＤ４５抗体、または抗Ｆｏｘｐ３抗体で３０分間染色した。Ｆｏｘｐ３染色は、Ｂｉｏｌｅｇｅｎｄの細胞内染色プロトコルに従って行った。その後、染色した細胞を、ＣＤ８およびＦｏｘｐ３細胞集団の分析のためにフローサイトメトリーに供した（分析のために２×１０4細胞を収集した）。 For T cell analysis, tumor tissue was excised 3 days after injection of aPD-L1, Gluc0-S-aPD-L1 and Gluc25-S-aPD-L1 and dissociated with BD Horizon ™ Dri Tissue & Tumor Dissociation Reagent. let me Tumor cells were then stained with anti-CD3, anti-CD8, anti-CD4, anti-CD45, or anti-Foxp3 antibodies for 30 minutes. Foxp3 staining was performed according to Biolegend's intracellular staining protocol. Stained cells were then subjected to flow cytometry for analysis of CD8 and Foxp3 cell populations (2×10 4 cells were collected for analysis).
　メモリーＴ細胞解析のために、生理食塩水およびＧｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１処理したマウスから６０日後に脾臓細胞を取り出し、抗ＣＤ８抗体、抗ＣＤ４４抗体および抗ＣＤ６２Ｌ抗体で染色した。その後、染色した細胞をフローサイトメトリーで解析した（解析のために２×１０4細胞を収集した）。 For memory T cell analysis, splenocytes were harvested from saline and Gluc25-S-aPD-L1 treated mice after 60 days and stained with anti-CD8, anti-CD44 and anti-CD62L antibodies. Stained cells were then analyzed by flow cytometry (2×10 4 cells were collected for analysis).
ＣＤ４５ + サブセット分析および正常臓器の炎症の亢進の検出
　未修飾ａＰＤ－Ｌ１とＧｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１を健康なマウス（ｎ＝８）に１０ｍｇ　ｋｇ-1の用量で週１回３回静脈内投与した。注射後５日目に肺、肝臓、および腎臓を摘出し、ＣＤ４５+細胞の分析を行った。一方、組織を１０．０μｍ切片に切断し、これを冷アセトンで固定し、ＰＢＳで洗浄し、室温で１時間、５％ＢＳＡでブロッキングした。その後、切片をＡｌｅｘａ　４８８標識抗ヒトＩｇＧ（Ｈ＋Ｌ）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ、カタログ番号Ａ－１１０１３）で４℃で一晩インキュベートし、その後、ＣＬＳＭ観察を行った。 CD45 + Subset Analysis and Detection of Increased Inflammation in Normal Organs Unmodified aPD-L1 and Gluc25-S-aPD-L1 were administered to healthy mice (n=8) at a dose of 10 mg kg −1 intravenously three times weekly. dosed. Five days after injection, lungs, livers and kidneys were harvested and analyzed for CD45 + cells. Meanwhile, the tissue was cut into 10.0 μm sections, which were fixed with cold acetone, washed with PBS, and blocked with 5% BSA for 1 hour at room temperature. Sections were then incubated with Alexa 488-labeled anti-human IgG (H+L) (Thermo Fisher Scientific, Catalog No. A-11013) overnight at 4° C., followed by CLSM observation.
　各組織のＴＮＦ－α、ＩＬ－６およびＩＬ－１βレベルをＥＬＩＳＡキット（サーモフィッシャー社製、カタログ番号ＢＭＳ６０７－３、ＫＭＣ００６１およびＢＭＳ６００２）を用いて測定した。肺、肝臓、および腎臓を含む組織は、指示書通りに各種処理を行った後、ホモジナイズし、５００ｇで１０分間遠心分離した。上清を分注し、分析まで－８０℃で保存した。サンプルはメーカーの指示に従いＥＬＩＳＡアッセイバッファーで希釈し、ＥＬＩＳＡキットを用いて分析した。
　Ａｌｅｘａ６４７標識ａＰＤ－Ｌ１およびＡｌｅｘａ６４７標識Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１をマウスに静脈内投与し、２４時間後に犠牲にして肺、肝臓および腎臓を抽出した。その後、組織を１０．０μｍ切片に切断し、これを冷アセトンで固定し、ＰＢＳで洗浄した。次いで、５％ＢＳＡで室温で１時間ブロッキングした。その後、切片をＡｌｅｘａ４８８標識抗ヒトＩｇＧ（Ｈ＋Ｌ）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ、カタログ番号Ａ－１１０１３）で４℃で一晩インキュベートし、その後、ＣＬＳＭ観察を行った。 TNF-α, IL-6 and IL-1β levels in each tissue were measured using ELISA kits (Thermo Fisher, catalog numbers BMS607-3, KMC0061 and BMS6002). Tissues including lung, liver, and kidney were treated according to instructions, then homogenized and centrifuged at 500 g for 10 minutes. Supernatants were aliquoted and stored at -80°C until analysis. Samples were diluted in ELISA assay buffer according to the manufacturer's instructions and analyzed using an ELISA kit.
Alexa647-labeled aPD-L1 and Alexa647-labeled Gluc25-S-aPD-L1 were administered intravenously to mice and sacrificed 24 hours later to extract lungs, livers and kidneys. Tissues were then cut into 10.0 μm sections, which were fixed with cold acetone and washed with PBS. It was then blocked with 5% BSA for 1 hour at room temperature. Sections were then incubated with Alexa488-labeled anti-human IgG (H+L) (Thermo Fisher Scientific, Catalog No. A-11013) overnight at 4° C., followed by CLSM observation.
統計
　すべての結果は、平均±ｓ．ｄ．または平均±ＳＥＭとして示されている。統計分析は、ＧｒａｐｈＰａｄ　Ｐｒｉｓｍ（８．０）を用いて評価した。生存時間の統計解析には対数順位検定を行い、その他の統計解析には、一方向分散分析（ＡＮＯＶＡ）に続いて、多重比較のためのテューキーのｈｏｎｅｓｔｌｙ　ｓｉｇｎｉｆｉｃａｎｔ　ｄｉｆｆｅｒｅｎｃｅ（ＨＳＤ）ポストホック検定を行った。実験群と対照群の間の差は、Ｐ　＜　０．０５で統計的に有意であると考えられた。＊Ｐ　＜　０．０５；　＊＊Ｐ　＜　０．０１；　および　＊＊＊Ｐ　＜　０．００１。 Statistics All results are mean ± sd. d. Or shown as mean ± SEM. Statistical analysis was evaluated using GraphPad Prism (8.0). Statistical analysis of survival time was performed by log-rank test, and other statistical analyzes were performed by one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) post-hoc test for multiple comparisons. . Differences between experimental and control groups were considered statistically significant at P < 0.05. *P <0.05; **P <0.01; and ***P < 0.001.
［結果］
還元環境応答性のＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１の調製と特性評価
　図１に示されるＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１を構築するために、まず、ＤＩＧ－ＰＥＧ－ＮＨ２またはα－ＣＨ３Ｏ－ＰＥＧ－ＮＨ２を過剰にして片側修飾を制御したＮＰＣ－（ＣＨ２）２－Ｓ－Ｓ－（ＣＨ２）２－ＮＰＣとＤＩＧ末端ポリエチレングリコール（ＤＩＧ－ＰＥＧ－ＮＨ２）またはα－メトキシ－ω－アミノポリエチレングリコール（α－ＭｅＯ－ＰＥＧ－ＮＨ２）との共有結合性のコンジュゲーション（図２参照）により、還元環境応答性のＰＥＧ鎖（ＤＩＧ－ＰＥＧ－Ｓ－ＮＰＣまたはＰＥＧ－Ｓ－ＮＰＣ）を合成し、その後、ＤＩＧをグルコースに脱保護した。１Ｈ核磁気共鳴（ＮＭＲ）スペクトルにポリマーの特徴的なピークを特定した。次に、Ｇｌｕｃ－ＰＥＧ－Ｓ－ＮＰＣとＰＥＧ－Ｓ－ＮＰＣの供給比率を調整して、グルコース濃度の異なる（０、２５、５０、１００モル％）一連のＧｌｕｃ－Ｓ－ＮＰＣを調製し、その後、共有結合法によりａＰＤ－Ｌ１のアミノ基と反応させた。その後、フルオレスカミンをプローブとしてａＰＤ－Ｌ１の残基アミノ基を検出したところ、約６０％のアミノ基が修飾されていることがわかった（図３）。 [result]
Preparation and characterization of reducing environment-responsive Gluc-S-aPD-L1 To construct Gluc-S - aPD -L1 shown in FIG. NPC-(CH 2 ) 2 -SS-(CH 2 ) 2 -NPC with excess PEG-NH 2 to control unilateral modification and DIG-terminated polyethylene glycol (DIG-PEG-NH 2 ) or α-methoxy- Covalent conjugation (see FIG. 2) with ω-aminopolyethylene glycol (α-MeO-PEG-NH 2 ) results in reducing environmentally responsive PEG chains (DIG-PEG-S-NPC or PEG-S- NPC) was synthesized, followed by deprotection of DIG to glucose. A characteristic peak of the polymer was identified in the 1 H nuclear magnetic resonance (NMR) spectrum. Next, by adjusting the feeding ratio of Gluc-PEG-S-NPC and PEG-S-NPC, a series of Gluc-S-NPCs with different glucose concentrations (0, 25, 50, 100 mol%) were prepared, Then, it was reacted with the amino group of aPD-L1 by a covalent bonding method. After that, when the residue amino groups of aPD-L1 were detected using fluorescamine as a probe, it was found that about 60% of the amino groups were modified (Fig. 3).
　さらに、抗体に対するＰＥＧ鎖の修飾と還元環境応答性の抗体からのＰＥＧ鎖の脱離を確認するために、まず動的光散乱（ＤＬＳ）解析を行い、サイズ分布とゼータ電位を評価した。図４左パネルに示すように、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１の流体力学的直径がわずかに増大したが、グルタチオン（ＧＳＨ）処理後にはＰＥＧ鎖切断を経て未修飾ａＰＤ－Ｌ１と同様に明瞭に減少することがわかった。なお、未修飾ａＰＤ－Ｌ１の体積平均粒径は１０．２ｎｍであり、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１の体積平均粒径は１８．８ｎｍであった。さらに、未修飾ａＰＤ－Ｌ１のゼータ電位は－６．１３ｍＶ、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１のゼータ電位は－０．１６ｍＶであったのに対し、グルタチオン（ＧＳＨ）処理では抗体からのＰＥＧ鎖の脱離が起こり、未修飾ａＰＤ－Ｌ１のゼータ電位とほぼ同等の－６．０７ｍＶの負電荷が得られた（図４右パネル）。このことから、図５に示されるように、ａＰＤ－Ｌ１は、アミノ酸側鎖のアミノ基においてグルコース修飾ＰＥＧ鎖で修飾されてＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１を形成すると、サイズが増大し、かつゼータ電位が上昇する。そして、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１は、還元環境下（例えば、ＧＳＨ存在下）では、ＰＥＧ鎖内のジスルフィド結合が開裂し、これにより未修飾形態のａＰＤ－Ｌ１に戻ることが示唆された。 Furthermore, in order to confirm the modification of the PEG chains on the antibody and the detachment of the PEG chains from the reducing-environment-responsive antibody, dynamic light scattering (DLS) analysis was first performed to evaluate the size distribution and zeta potential. As shown in FIG. 4, left panel, the hydrodynamic diameter of Gluc-S-aPD-L1 increased slightly, but after glutathione (GSH) treatment, via PEG chain scission, it became apparent similar to unmodified aPD-L1. found to decrease. The volume average particle size of unmodified aPD-L1 was 10.2 nm, and the volume average particle size of Gluc-S-aPD-L1 was 18.8 nm. Furthermore, unmodified aPD-L1 had a zeta potential of −6.13 mV and Gluc-S-aPD-L1 had a zeta potential of −0.16 mV, whereas glutathione (GSH) treatment resulted in a reduction of PEG chains from the antibody. Elimination occurred, resulting in a negative charge of −6.07 mV, which is almost the same as the zeta potential of unmodified aPD-L1 (Fig. 4 right panel). Thus, as shown in FIG. 5, aPD-L1 increases in size and zeta- potential rises. It was also suggested that Gluc-S-aPD-L1 reverts to the unmodified form of aPD-L1 by cleaving the disulfide bond in the PEG chain under a reducing environment (eg, in the presence of GSH).
　さらに、ゲル浸透クロマトグラフィー（ＧＰＣ）を用いて、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１がＰＥＧ化処理後に明らかに分子量が増加していることを示した。一方、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１に関連するピークは、還元環境下では、時間の経過とともに徐々に元のａＰＤ－Ｌ１に戻ることがわかり（図６）、還元環境応答性のＰＥＧ鎖の切断挙動をさらに明らかにした。さらに、我々は、円二色性スペクトル24を使用して、ＰＥＧ鎖の導入と開裂後のａＰＤ－Ｌ１の二次構造を分析した。ＧＳＨ処理の有無にかかわらず、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１は未修飾ａＰＤ－Ｌ１と比較して有意差のないβシート吸光度を示したことから、構造がよく保存されたＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１は、ＰＥＧ鎖切断後もＰＤ－Ｌ１に対して生理機能を維持していることが示唆された25。二次構造を除いては、応答性のＰＥＧ鎖切断後もリジンのアミン基を占有する残基を含まないａＰＤ－Ｌ１の表面構造が保存されていることから、生理機能が維持されていることがさらに確認された（図５）。
　Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１のリガンドに対する結合親和性を検証するために、酵素結合免疫吸着法（ＥＬＩＳＡ）26を用いて、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１のリガンドに対する特異的な結合能を定量した。図６右に示されるように、ＥＬＩＳＡでは、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１は、ＰＤ－Ｌ１に対して有意な結合を示さなかった。これに対して、還元環境下（１．０　ｍＭ　ＧＳＨ、ｐＨ　７．４）にＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１を曝露すると、時間依存的に抗体の結合量が有意に増加した（２時間以上のインキュベーションでほぼプラトーに達する）ことから、還元環境下ではＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１のジスルフィド結合が切断されて、未修飾のａＰＤ－Ｌ１が放出されたことが示唆された27。さらに、様々なＰＥＧ鎖の密度を有するａＰＤ－Ｌ１を調製し、その結合親和性を評価したところ、ａＰＤ－Ｌ１表面の修飾アミン基の割合が少ない場合には、ＰＤ－Ｌ１に対する結合能が部分的に残存した。 Furthermore, gel permeation chromatography (GPC) was used to show that Gluc-S-aPD-L1 clearly increased in molecular weight after PEGylation treatment. On the other hand, it was found that the peak associated with Gluc-S-aPD-L1 gradually returned to the original aPD-L1 over time under a reducing environment (Fig. 6), suggesting that the PEG chain cleavage responsive to the reducing environment behavior is further clarified. In addition, we used circular dichroism spectra24 to analyze the secondary structure of aPD-L1 after introduction and cleavage of PEG chains. With or without GSH treatment, Gluc-S-aPD-L1 showed no significant difference in β-sheet absorbance compared to unmodified aPD-L1, suggesting that Gluc-S-aPD-L1 with well-preserved structure It was suggested that L1 maintains physiological function to PD-L1 after PEG chain cleavage 25 . Except for the secondary structure, the surface structure of aPD-L1, which does not contain residues occupying the amine groups of lysines, is preserved after responsive PEG chain cleavage, thus maintaining physiological function. was further confirmed (Fig. 5).
To verify the binding affinity of Gluc-S-aPD-L1 to its ligands, an enzyme-linked immunosorbent assay (ELISA) 26 was used to quantify the specific binding ability of Gluc-S-aPD-L1 to its ligands. . As shown in FIG. 6, right, Gluc-S-aPD-L1 did not show significant binding to PD-L1 in ELISA. In contrast, when Gluc-S-aPD-L1 was exposed to a reducing environment (1.0 mM GSH, pH 7.4), the antibody binding amount increased significantly in a time-dependent manner (2 hours or longer). incubation almost reached a plateau), suggesting that the disulfide bond of Gluc-S-aPD-L1 was cleaved to release unmodified aPD-L1 under the reducing environment 27 . Furthermore, aPD-L1 with various densities of PEG chains was prepared and its binding affinity was evaluated. effectively survived.
　ＰＤ－Ｌ１は主に腫瘍細胞の膜に発現している28ので、ＧＬ２６１神経膠芽腫細胞に対するＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１の特異的な結合を評価した。ポジティブコントロールとして未修飾のａＰＤ－Ｌ１は、１．０時間のインキュベーション後に腫瘍細胞膜との好ましい共局在化を示したが、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１と腫瘍細胞とのインキュベーション後には、無視できる程度の赤色蛍光しか認められなかった（図７）。これは、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１では、ＰＥＧ鎖による修飾のために抗体がＰＤ－Ｌ１との結合性を大きく損なっているためであると考えられる。これに対して、還元環境下で処理されたＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１は、抗体からのＰＥＧ鎖の効率的な切断に伴う未修飾型のａＰＤ－Ｌ１の増加により、ＰＤ－Ｌ１への結合親和性を明らかに回復させた（図６および図７）。そしてこのことは、フローサイトメトリー解析によるＧＬ２６１細胞のＰＤ－Ｌ１発現の定量的な測定によってさらに実証された。以上の結果から、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１は、ＰＥＧ修飾によりその結合親和性を大きく損なっているが、還元環境下ではＰＥＧ鎖の切断と未修飾型のａＰＤ－Ｌ１の放出により、結合能力を回復することが確認された。体内では、脳実質は還元環境であることが知られ、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１は、脳実質特異的に結合親和性を回復させ、脳における部位特異的なＩＣＢ療法（特に、他臓器では抗体は非活性である）を可能とすると考えられた。 Since PD-L1 is primarily expressed on the membrane of tumor cells28 , the specific binding of Gluc-S-aPD-L1 to GL261 glioblastoma cells was assessed. Unmodified aPD-L1 as a positive control showed favorable co-localization with tumor cell membranes after 1.0 hour incubation, but negligible after incubation of Gluc-S-aPD-L1 with tumor cells. Only moderate red fluorescence was observed (Fig. 7). This is probably because Gluc-S-aPD-L1 significantly impairs the binding of the antibody to PD-L1 due to modification with a PEG chain. In contrast, Gluc-S-aPD-L1 treated in a reducing environment binds to PD-L1 due to an increase in unmodified aPD-L1 following efficient cleavage of the PEG chain from the antibody. Affinity was clearly restored (Figs. 6 and 7). And this was further substantiated by quantitative measurement of PD-L1 expression in GL261 cells by flow cytometric analysis. These results suggest that PEG modification of Gluc-S-aPD-L1 greatly impairs its binding affinity, but under a reducing environment, PEG chain cleavage and release of unmodified aPD-L1 reduce the binding affinity. was confirmed to restore In vivo, the brain parenchyma is known to be a reducing environment, and Gluc-S-aPD-L1 restores binding affinity specifically in the brain parenchyma, and is effective for site-specific ICB therapy in the brain (especially in other organs). antibody is inactive).
Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１のｉｎ　ｖｉｖｏ治療効果
　従来の神経膠芽腫（ＧＢＭ）治療の失敗の主な原因の一つは、血液脳関門（ＢＢＢ）の存在による治療薬の脳への送達効率の低さである2,3。そのため、ＧＢＭ部位へのＩＣＢ抗体の選択的な集積を促進することが治療指標の向上に重要であると考えられる。Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１のＢＢＢ輸送能を調べるために、我々はまず、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１のｉｎ　ｖｉｖｏ薬物動態を評価した。図８左パネルに示すように、様々なグルコース密度を有するＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１は、未修飾のａＰＤ－Ｌ１（ｔ1/2β＝５．２時間）よりも有意に長い半減期（ｔ１／２β＝６．１～１０．３時間）を示したが、これはコアに対するＰＥＧシェルコーティングが血中安定性を高め、かつ、オフターゲット結合を介して正常臓器への分布を抑制したことに起因する可能性がある23。さらに、より高いグルコース密度の導入は、肝臓で発現するＧＬＵＴファミリーとの相互作用が強化されているため、比較的短い血液循環を引き起こす29,30。そして、ａＰＤ－Ｌ１のグルコース導入密度を調節することは、腫瘍の蓄積を強化するための循環を延長するために非常に有利であることを示唆している。次に、同所性ＧＢＭモデルを用いてＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１のＢＢＢの能動的な輸送能力を試験した。ａＰＤ－Ｌ１をまず蛍光色素で標識し、その後、Ｇｌｕｃ－ＰＥＧ－Ｓ－ＮＰＣとＰＥＧ－Ｓ－ＮＰＣをそれぞれ修飾した。未修飾のａＰＤ－Ｌ１および修飾グルコース密度を変化させたＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１を、同等のａＰＤ－Ｌ１用量で同所性ＧＢＭ腫瘍を有するＣ５７ＢＬ／６Ｊマウスに静脈内注射し、主要臓器を注射後２４時間の間に蛍光分析のために取り出した。明らかに、未修飾のａＰＤ－Ｌ１とコントロールとしての活性なターゲティング能力を持たないＧｌｕｃ０－Ｓ－ａＰＤ－Ｌ１は２４時間の間に脳内の腫瘍への蓄積が認められたがその量は少なく、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１は２．２％のＩＤ　ｇ－１で最も高い蓄積量を示し、初期４時間ではＧｌｕｃ０－Ｓ－ａＰＤ－Ｌ１群よりも～６．１倍、未修飾のａＰＤ－Ｌ１群よりも～１８．０倍の増加した蓄積量を示した（図９左パネル）。このことは、抗体は、修飾グルコースによって、ＢＢＢ通過の能動的輸送能を高めたことを示す31-33。一方、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１は、肝臓、脾臓、腎臓、肺にも蓄積しており、グルコース密度が高く導入されたａＰＤ－Ｌ１は肝臓に取り込まれやすく、Ｇｌｕｃ５０－Ｓ－ａＰＤ－Ｌ１とＧｌｕｃ１００－Ｓ－ａＰＤ－Ｌ１の血行が低下していることと相関している。これらの現象は、肝臓の血管内コンパートメントに存在する様々なＧＬＵＴに起因する可能性があり、表面上の適切なグルコース密度は、肝臓の障壁を克服し、腫瘍蓄積31を高めるための鍵であることを示唆する。さらに興味深いことに、ＧＬＵＣ２５－Ｓ－ａＰＤ－Ｌ１のＢＢＢを通過する能動的輸送機構を伴う迅速な送達により、脳腫瘍部位に選択的に集積し、腫瘍／脳の比率は最大４５（図９右パネル）まで上昇した。これは、本発明の技術が、脳における免疫チェックポイント阻害剤（ＩＣＢ）療法において有効であることを示唆する。なお、本実験は、特別な血糖操作（特に絶食後に血糖値上昇操作と共に抗体を投与するなどの血糖操作）は実施していない。脳腫瘍を有する対象においてはＢＢＢが破壊されており、薬物が脳腫瘍に到達し得る。
　同所性脳腫瘍に対するＧｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１の治療効果を調べるために、ＧＬ２６１細胞株を脳の右側の頭蓋内に接種した。その後、ＧＬ２６１腫瘍を有するマウスに生理食塩水、ａＰＤ－Ｌ１、Ｇｌｕｃ０－Ｓ－ａＰＤ－Ｌ１、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１を１．５ｍｇ／ｋｇの用量で単回投与した。一方、不活性化型（非開裂型）のＧｌｕｃ２５－Ｃ－ａＰＤ－Ｌ１を対照として適用した。脳腫瘍の発生は、全脳におけるＧＬ２６１細胞の生物発光によって追跡された。図１０－１に示すように、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１で処理したマウスは、６日後に生物発光が減少し、５匹のマウスのうち２匹は、脳部位で無視できるレベルのＧＬ２６１細胞しか検出されず、このことは強い免疫応答を示唆する。対照的に、未修飾のａＰＤ－Ｌ１およびＧｌｕｃ０－Ｓ－ａＰＤ－Ｌ１を投与されたマウスでは、ＧＬ２６１腫瘍に対して弱い治療効果しか得られなかった。このように治療効果は脳内への抗体の蓄積量に応じたものであった。さらに、不活性化型（非開裂型）Ｇｌｕｃ２５－Ｃ－ａＰＤ－Ｌ１を投与したマウスのＧＬ２６１腫瘍もまた強い増殖プロファイルを示したが、これは不活性化型（非開裂型）Ｇｌｕｃ２５－Ｃ－ａＰＤ－Ｌ１が還元に反応してａＰＤ－Ｌ１を放出しないことに起因するものであり、還元環境と還元環境応答性のリンカーの存在が本発明の脳指向性のＩＣＢ治療に重要な役割を果たしていることを示唆している（図１０－１下の５つのパネル）。Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１を投与したマウスでは、９０日後の生存率は約６０％であったが、３２日以上生存したマウスはなく、他のすべての対照群で明らかな体重減少が認められた（図１０－２）。ＧＬ２６１に対する強力な抗癌効果に鑑みて、我々はさらにＧｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１をＣＴ－２Ａ細胞株の治療に適用した。ＣＴ－２Ａ細胞株は、ＰＴＥＮの低発現レベルを示し34、腫瘍内不均一性を含む、臨床的神経膠芽腫のいくつかの特徴を正確に反映しており、かつ、高比率のがん幹細胞の存在のために放射線耐性および化学療法耐性を示す34,35。生理食塩水投与群のＣＴ－２Ａ腫瘍を有するマウスは、生存期間が２０日未満で積極的な腫瘍増殖を示した（図１１）。未修飾のａＰＤ－Ｌ１投与群でも同様であった。これに対して、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１を投与したマウスでは、腫瘍の成長を顕著に遅らせ、生存期間を延長した（図１１）。このことは、ＢＢＢを介したＩＣＢ抗体の積極的な送達と再活性化可能なＩＣＢ療法の両方が、十分かつ強固な抗腫瘍免疫応答を介して、侵攻性の高い神経膠芽腫に対する治療効果を高めることを示している。 In Vivo Therapeutic Efficacy of Gluc-S-aPD-L1 One of the major causes of failure of conventional glioblastoma (GBM) treatments is the inefficiency of delivery of therapeutics to the brain due to the presence of the blood-brain barrier (BBB). 2,3 which is low. Therefore, promoting the selective accumulation of ICB antibodies at the GBM site is considered important for improving the therapeutic index. To investigate the BBB transport ability of Gluc-S-aPD-L1, we first evaluated the in vivo pharmacokinetics of Gluc-S-aPD-L1. As shown in FIG. 8 left panel, Gluc-S- aPD -L1 with different glucose densities has a significantly longer half-life ( t 1/2 β = 6.1-10.3 hours), indicating that the PEG shell coating on the core enhances blood stability and inhibits distribution to normal organs via off-target binding. 23 Furthermore, introduction of higher glucose densities causes relatively short blood circulation due to enhanced interactions with the GLUT family expressed in the liver 29,30 . This suggests that modulating the glucose uptake density of aPD-L1 is highly beneficial for prolonging circulation to enhance tumor accumulation. Next, the ability of Gluc-S-aPD-L1 to actively transport across the BBB was tested using the orthotopic GBM model. aPD-L1 was first labeled with a fluorescent dye and then modified with Gluc-PEG-S-NPC and PEG-S-NPC, respectively. Unmodified aPD-L1 and Gluc-S-aPD-L1 with varying modified glucose densities were injected i.v. Removed for fluorescence analysis during 24 hours post-injection. Clearly, unmodified aPD-L1 and Gluc0-S-aPD-L1, which does not have active targeting ability as a control, accumulated in tumors in the brain during 24 hours, but the amount was small. Gluc25-S-aPD-L1 showed the highest accumulation with an ID g- 1 of 2.2%, ~6.1-fold higher than the Gluc0-S-aPD-L1 group in the first 4 hours, and unmodified aPD-L1 It showed an increased amount of accumulation of ~18.0-fold over the L1 group (Fig. 9 left panel). This indicates that the antibody enhanced active transport across the BBB with modified glucose 31-33 . On the other hand, Gluc-S-aPD-L1 is also accumulated in the liver, spleen, kidney, and lungs, and aPD-L1 introduced with high glucose density is easily taken up by the liver, and Gluc50-S-aPD-L1 and It correlates with decreased blood circulation of Gluc100-S-aPD-L1. These phenomena may be attributed to the various GLUTs present in the intravascular compartment of the liver, and adequate glucose density on the surface is key to overcoming the liver barrier and enhancing tumor accumulation. suggest that More interestingly, rapid delivery of GLUC25-S-aPD-L1 with an active transport mechanism across the BBB resulted in selective accumulation at brain tumor sites, with tumor/brain ratios up to 45 (Fig. 9 right panel). ). This suggests that the technology of the present invention is effective in immune checkpoint inhibitor (ICB) therapy in the brain. In this experiment, no special blood sugar manipulation (particularly, blood sugar manipulation such as administering an antibody together with a blood sugar level increasing manipulation after fasting) was performed. The BBB is disrupted in subjects with brain tumors, allowing drugs to reach the brain tumor.
To examine the therapeutic effect of Gluc25-S-aPD-L1 on orthotopic brain tumors, the GL261 cell line was inoculated intracranially on the right side of the brain. GL261 tumor-bearing mice were then given a single dose of saline, aPD-L1, Gluc0-S-aPD-L1, Gluc25-S-aPD-L1 at a dose of 1.5 mg/kg. On the other hand, inactivated (uncleaved) Gluc25-C-aPD-L1 was applied as a control. Brain tumor development was followed by bioluminescence of GL261 cells in the whole brain. As shown in FIG. 10-1, mice treated with Gluc25-S-aPD-L1 had reduced bioluminescence after 6 days, with 2 out of 5 mice having negligible levels of GL261 cells in brain regions. was detected, suggesting a strong immune response. In contrast, mice receiving unmodified aPD-L1 and Gluc0-S-aPD-L1 had only weak therapeutic effects against GL261 tumors. Thus, the therapeutic effect was dependent on the amount of antibody accumulated in the brain. Furthermore, GL261 tumors from mice treated with inactivated (uncleaved) Gluc25-C-aPD-L1 also showed a robust growth profile, which was consistent with inactivated (uncleaved) Gluc25-C-aPD-L1. This is due to the fact that aPD-L1 does not release aPD-L1 in response to reduction, and the presence of a reducing environment and a linker responsive to the reducing environment plays an important role in the brain-directed ICB therapy of the present invention. (Fig. 10-1, bottom five panels). Mice treated with Gluc25-S-aPD-L1 had a survival rate of approximately 60% after 90 days, but no mice survived more than 32 days, and all other control groups showed significant weight loss. (Fig. 10-2). Given the potent anti-cancer effect on GL261, we also applied Gluc25-S-aPD-L1 to treat the CT-2A cell line. The CT-2A cell line exhibits low levels of PTEN expression34, accurately reflecting several features of clinical glioblastoma , including intratumoral heterogeneity, and a high proportion of cancers. Due to the presence of stem cells, it is radio- and chemo-resistant 34,35 . CT-2A tumor-bearing mice in the saline-treated group showed aggressive tumor growth with a survival time of less than 20 days (FIG. 11). The same was true for the unmodified aPD-L1 administration group. In contrast, mice treated with Gluc25-S-aPD-L1 significantly retarded tumor growth and prolonged survival (FIG. 11). This suggests that both aggressive delivery of ICB antibodies via the BBB and reactivatable ICB therapy can be therapeutically effective against highly aggressive glioblastoma through a robust and robust anti-tumor immune response. increase the
Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１の生体内ＰＥＧ鎖切断
ＧＳＨとアスコルビン酸（ビタミンＣとも呼ばれる）は、脳の酸化ストレスに対する中枢神経系の最も強力な還元性因子と考えられており、そのレベルは約ミリモルであり36,37、正常な機能の維持と神経細胞の生存に不可欠である。還元によって生じるＰＥＧ鎖の切断を直接観察するために、Ａｌｅｘａ４８８標識の抗ヒト二次抗体を用いて、Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１と還元に応答して放出されたａＰＤ－Ｌ１を区別することにした。Ａｌｅｘａ４８８標識の抗ヒト二次抗体は、放出されたａＰＤ－Ｌ１には結合するが、ＰＥＧ鎖のシェル構造のためにＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１には結合を示さない（図１２）。ａＰＤ－Ｌ１を最初にＡｌｅｘａ６４７色素で標識し、次にＧｌｕｃ０－Ｓ－ａＰＤ－Ｌ１、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１およびＧｌｕｃ２５－Ｃ－ａＰＤ－Ｌ１をそれぞれ調製した。次に、各種製剤を同所性脳腫瘍を有するマウスに静脈内注射した。２４時間後にマウスを犠牲にして腫瘍組織を取り出した。取り出した組織から組織切片を作製し、抗ヒト二次抗体でインキュベートした。図１３に示すように、ＢＢＢ能力を横切る活性輸送を持たない未修飾のａＰＤ－Ｌ１は、腫瘍部位での蓄積が最小限であるのに対し、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１およびＧｌｕｃ２５－Ｃ－ａＰＤ－Ｌ１は、グルコースの導入により高い脳内分布を示した。さらに、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１は、還元環境に応答してＰＥＧ鎖が効率的に切断され、ａＰＤ－Ｌ１が放出されることを明らかにした（図１３左下パネル）。また、緑色蛍光で標識されたＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１は、ＰＥＧ鎖切断の有無にかかわらず、全Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１と７２％の共局在化を示し、還元的シグナルを感知した際に７２％のＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１のＰＤ－Ｌ１に対する結合能が回復したことを反映していると考えられる。一方、Ｇｌｕｃ２５－Ｃ－ａＰＤ－Ｌ１で処理したマウスでは、ＰＥＧ鎖の切断が困難なため、無視できる程度のＡｌｅｘａ４８８の蛍光しか示さなかった（図１３右下パネル）。したがって、ＰＥＧ鎖切断を介したＧｌｕｃ－Ｓ－ａＰＤ－Ｌ１の還元環境依存的なａＰＤ－Ｌ１放出挙動は、不活性化された抗体による脳組織以外での副作用の軽減と、脳内における再活性化に基づくＩＣＢ療法の途を拓くものである。 In vivo PEG chain scission of Gluc-S-aPD-L1 GSH and ascorbic acid (also called vitamin C) are considered the most potent reducing factors of the central nervous system against oxidative stress in the brain, with levels of approximately It is millimolar 36,37 and is essential for the maintenance of normal function and survival of neurons. To directly observe the PEG chain cleavage that occurs upon reduction, we used an Alexa488-labeled anti-human secondary antibody to distinguish between Gluc-S-aPD-L1 and aPD-L1 released in response to reduction. did. An Alexa488-labeled anti-human secondary antibody binds to released aPD-L1 but shows no binding to Gluc-S-aPD-L1 due to the shell structure of the PEG chains (FIG. 12). aPD-L1 was first labeled with Alexa647 dye, then Gluc0-S-aPD-L1, Gluc25-S-aPD-L1 and Gluc25-C-aPD-L1 were prepared respectively. Various formulations were then injected intravenously into mice bearing orthotopic brain tumors. Twenty-four hours later, mice were sacrificed and tumor tissue was removed. Tissue sections were made from the removed tissue and incubated with anti-human secondary antibody. As shown in FIG. 13, unmodified aPD-L1, which has no active transport across the BBB capacity, has minimal accumulation at tumor sites, whereas Gluc25-S-aPD-L1 and Gluc25-C- aPD-L1 showed high brain distribution upon introduction of glucose. Furthermore, Gluc25-S-aPD-L1 revealed efficient PEG chain cleavage in response to a reducing environment, releasing aPD-L1 (FIG. 13, lower left panel). Green fluorescence-labeled Gluc-S-aPD-L1 also showed 72% co-localization with total Gluc-S-aPD-L1 with or without PEG strand scission, sensing reductive signals. This is thought to reflect the recovery of 72% of the ability of Gluc-S-aPD-L1 to bind to PD-L1 when the strain was applied. On the other hand, mice treated with Gluc25-C-aPD-L1 showed negligible Alexa488 fluorescence due to difficulty in PEG chain cleavage (FIG. 13, lower right panel). Therefore, the reductive environment-dependent aPD-L1 release behavior of Gluc-S-aPD-L1 via PEG chain scission is expected to reduce the side effects of the inactivated antibody outside the brain tissue and reactivate it in the brain. This will open the way for ICB therapy based on biochemistry.
Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１による免疫応答の誘導
Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１の治療効果の背景にある免疫応答を調べるために、注射後３日目に腫瘍内のＴ細胞を採取し、フローサイトメトリーで分析した。腫瘍細胞を攻撃するための優勢なＴ細胞集団と考えられる細胞傷害性Ｔ細胞（ＣＤ８＋Ｔ細胞）の数は、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１投与群では、生理食塩水群、ａＰＤ－Ｌ１投与群、Ｇｌｕｃ０－Ｓ－ａＰＤ－Ｌ１投与群と比較して２～３倍の増加を示した（図１４－１）。このことは、本発明の腫瘍標的化と脳内での抗体の再活性化の戦略が、頑健な抗腫瘍免疫応答の誘導に有効であることを示す。浸潤ＣＤ８＋Ｔ細胞の増加は、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１処理マウスの腫瘍切片の免疫染色によっても証明された（図１４－２）。それに加えて、さらに、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１を投与されたマウスにおいて、抗原のプロセッシングおよび提示メカニズムを刺激することによってＴ細胞応答を典型的に促進するＩＦＮγの放出を試験したところ、他の対照群に比べて２～３倍高い値を示した（図１４－３）。さらに、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１投与群では、免疫抑制性のＦｏｘｐ３＋Ｔ細胞（制御性Ｔ細胞、Ｔｒｅｇ）の集団が対照群と比較して明らかに減少しており（図１５）、免疫抑制性の微小環境を緩和することで、免疫治療効果を高めることが可能であることが示唆された。このことから、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１は再活性化可能な免疫療法剤として作用し、ＣＤ８＋Ｔ細胞の浸潤を促進し、腫瘍部位でのＴｒｅｇを低下させることで免疫応答を高めることができ、抗腫瘍効果が期待できると考えられた。
　Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１を投与されたマウスにおいて、腫瘍再発の効果的な予防を促進するメモリーＴ細胞のプライミングを検証するために、脾臓のＴ細胞を採取し、フローサイトメトリーを用いて分析した。Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１投与群では、ネイティブマウスと比較してＣＤ４４ｈｉＣＤ６２ＬｌｏｗエフェクターメモリーＴ細胞サブセットが約２．０倍に増加していることが確認された（図１６）。エフェクターメモリーＴ細胞の機能は、腫瘍再チャレンジ実験によりさらに確認された。Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１を脳の右側に投与して腫瘍を消失させたマウスに、再びＧＬ２６１腫瘍細胞を脳の左側で再チャレンジさせたところ（図１７－１）、図１７－２に示されるように、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１投与群では、無腫瘍状態を維持したまま腫瘍の増殖が抑制されたのに対し、天然マウスは２５日以内の生存期間で急速に腫瘍が増殖することが確認された。これらの知見は、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１が長期生存のために腫瘍の再発を効果的に防止できることを示唆している。 Induction of Immune Response by Gluc-S-aPD-L1 In order to investigate the immune response behind the therapeutic effect of Gluc25-S-aPD-L1, intratumoral T cells were collected 3 days after injection and analyzed by flow cytometry. analyzed by metric. The number of cytotoxic T cells (CD8 + T cells), which is considered to be the predominant T cell population for attacking tumor cells, was higher in the Gluc25-S-aPD-L1 administration group than in the saline group, the aPD-L1 administration group, It showed a 2- to 3-fold increase compared to the Gluc0-S-aPD-L1 administration group (Fig. 14-1). This indicates that the tumor targeting and antibody reactivation strategies in the brain of the present invention are effective in inducing robust anti-tumor immune responses. An increase in infiltrating CD8+ T cells was also demonstrated by immunostaining of tumor sections from Gluc25-S-aPD-L1 treated mice (Fig. 14-2). In addition, we further tested the release of IFNγ, which typically promotes T cell responses by stimulating antigen processing and presentation mechanisms, in mice administered Gluc25-S-aPD-L1, and found that other The values were 2 to 3 times higher than those of the control group (Fig. 14-3). Furthermore, in the Gluc25-S-aPD-L1 administration group, the population of immunosuppressive Foxp3 + T cells (regulatory T cells, Treg) was clearly reduced compared to the control group (Fig. 15), indicating that immunosuppressive It was suggested that it is possible to enhance the immunotherapeutic effect by relaxing the microenvironment of the cell. This suggests that Gluc25-S-aPD-L1 can act as a reactivatable immunotherapeutic agent, enhancing the infiltration of CD8+ T cells and increasing the immune response by reducing Tregs at tumor sites. It was thought that an antitumor effect could be expected.
To verify the priming of memory T cells that promotes effective prevention of tumor recurrence in mice treated with Gluc25-S-aPD-L1, splenic T cells were harvested and analyzed using flow cytometry. did. In the Gluc25-S-aPD-L1 administration group, it was confirmed that the CD44hiCD62Llow effector memory T cell subset increased approximately 2.0-fold compared to native mice (Fig. 16). The function of effector memory T cells was further confirmed by tumor rechallenge experiments. Mice in which Gluc25-S-aPD-L1 was administered on the right side of the brain to cause tumor disappearance were re-challenged with GL261 tumor cells on the left side of the brain (FIG. 17-1), as shown in FIG. 17-2. As shown, in the Gluc25-S-aPD-L1 administration group, tumor growth was suppressed while maintaining a tumor-free state, whereas natural mice showed rapid tumor growth within a survival period of 25 days. was confirmed. These findings suggest that Gluc25-S-aPD-L1 can effectively prevent tumor recurrence for long-term survival.
Ｇｌｕｃ－Ｓ－ａＰＤ－Ｌ１によるｉｒＡＥの発生抑制
　血管内皮壁に発現しているＰＤ－Ｌ１のために、従来技術においては、ａＰＤ－Ｌ１の最初のオフターゲットプロセスは血液循環中で生じ、これが全身性炎症を誘発すると考えるのが合理的である26。そこで、２４時間以内にＧｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１から放出されたａＰＤ－Ｌ１を血流中で定量的に測定した。その結果、多量の未修飾のａＰＤ－Ｌ１はＰＤ－Ｌ１に対する本来の結合親和性を示したが、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１群では生理的環境下でのオフターゲット結合をさらに回避したことが判明した。血中での還元レベルが限られているため、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１から放出されたａＰＤ－Ｌ１の量はわずかであり、ａＰＤ－Ｌ１のＰＥＧ化戦略が、抗体の血管内皮壁への結合親和性を効果的にブロックしたことを示唆している（図８右パネル）。
　さらに、オフターゲット効果の低減を実証するために、非標的組織（肺、肝臓、腎臓など）を選択し、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１を用いて免疫関連性の有害事象（ｉｒＡＥ）を抑制する可能性を評価した。オフターゲット効果に起因する有害事象は、非標的組織の実質内で免疫系が過剰に活性化され、リンパ球の浸潤や炎症性サイトカインの産生が伴うと広く予測されている11-13。未修飾のａＰＤ－Ｌ１およびＧｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１の５日後の注射で、肺、および腎臓は、浸潤リンパ球（ＣＤ４５＋細胞）および炎症促進性サイトカイン（例えば、ＴＮＦ－α、ＩＬ－６およびＩＬ－１β）の放出の測定のために摘出された。図１８に示すように、ＣＤ４５＋細胞の浸潤の有意な改善は、未修飾のａＰＤ－Ｌ１を受けたマウスの一部で明らかに検出されたが、これは、自己免疫活性化を誘導するための正常組織におけるオフターゲット結合親和性に起因すると考えられる。対照的に、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１投与群のマウスは、生理食塩水投与群とほぼ同等の比較的低いリンパ球浸潤しか示さず、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１の非活性化機能が非標的組織におけるリンパ球の過剰浸潤のリスク低下に寄与していることが示唆された。さらに、組織をホモジナイズし、非標的組織内で放出された炎症性サイトカインの動態を調べたところ38、生理食塩水投与群やＧｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１投与群に比べ、未修飾のａＰＤ－Ｌ１を投与したマウスの２５％のＴＮＦ－α、ＩＬ－６、ＩＬ－１βレベルが２～４倍と異常に上昇していることがわかった（図１９）。さらに、組織切片を染色して、非標的組織においてＧｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１からａＰＤ－Ｌ１が放出され得るかどうかを検討した（図２０）。対照群のＡｌｅｘａ　６４７で標識された未修飾のａＰＤ－Ｌ１は、緑色蛍光との共局在化を伴って非標的組織に分布しており、ＰＤ－Ｌ１に対する結合親和性が、正常臓器で発現したＰＤ－Ｌ１へのオフターゲット結合を示し、これにより自己免疫活性化に明確に起因していることを示唆している（図２０下パネル）。しかし、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１は、非標的組織においては、還元環境の欠如により、ａＰＤ－Ｌ１の放出に失敗し、非標的組織中のＰＤ－Ｌ１の標的化ができなかった。このことが、Ｇｌｕｃ２５－Ｓ－ａＰＤ－Ｌ１が非標的組織中では、免疫活性化を誘導しなかった理由であると考えられる。以上のことから、非標的組織での有害事象は、脳組織特異的に再活性化可能な本発明の抗体を用いたＩＣＢ治療を行うことで回避でき、副作用を抑制しながら臨床成績を向上させることが可能であることが示唆された。 Suppression of irAE Occurrence by Gluc-S-aPD-L1 Due to PD-L1 being expressed on the vascular endothelial wall, in the prior art, the first off-target process of aPD-L1 occurs in the blood circulation, which is systemic. It is reasonable to assume that it induces sexual inflammation26 . Therefore, aPD-L1 released from Gluc25-S-aPD-L1 within 24 hours was quantitatively measured in the bloodstream. As a result, it was shown that while high amounts of unmodified aPD-L1 exhibited native binding affinity for PD-L1, the Gluc25-S-aPD-L1 group further avoided off-target binding under physiological conditions. found. Due to limited levels of reduction in the blood, the amount of aPD-L1 released from Gluc25-S-aPD-L1 was insignificant, and the aPD-L1 PEGylation strategy was effective in enhancing the delivery of antibodies to the vascular endothelial wall. It suggests that it effectively blocked the binding affinity (Fig. 8 right panel).
Additionally, to demonstrate reduced off-target effects, select non-target tissues (lung, liver, kidney, etc.) to suppress immune-related adverse events (irAEs) using Gluc25-S-aPD-L1 evaluated the possibilities. Adverse events resulting from off-target effects are widely predicted to be hyperactivation of the immune system within the parenchyma of non-target tissues, accompanied by lymphocyte infiltration and production of inflammatory cytokines 11-13 . Five days post-injection of unmodified aPD-L1 and Gluc25-S-aPD-L1, lungs and kidneys were enriched with infiltrating lymphocytes (CD45+ cells) and pro-inflammatory cytokines (e.g., TNF-α, IL-6 and IL-1β) were excised for measurement of release. As shown in FIG. 18, a significant improvement in CD45+ cell infiltration was clearly detected in some of the mice that received unmodified aPD-L1, suggesting that cytotoxicity is an important factor for inducing autoimmune activation. It is thought to be due to off-target binding affinities in normal tissues. In contrast, mice in the Gluc25-S-aPD-L1-administered group showed relatively low lymphocyte infiltration, almost equivalent to the saline-administered group, indicating that the non-activating function of Gluc25-S-aPD-L1 was non-existent. It was suggested that it contributes to the reduction of the risk of excessive infiltration of lymphocytes in the target tissue. Furthermore, when the tissue was homogenized and the kinetics of inflammatory cytokines released in non-target tissues was examined, 38 unmodified aPD-L1 was significantly more active than saline-treated or Gluc25-S-aPD-L1-treated groups. It was found that TNF-α, IL-6 and IL-1β levels in 25% of the mice treated with 2- to 4-fold abnormally increased (Fig. 19). In addition, tissue sections were stained to investigate whether aPD-L1 could be released from Gluc25-S-aPD-L1 in non-target tissues (FIG. 20). Control Alexa 647-labeled unmodified aPD-L1 was distributed in non-target tissues with co-localization with green fluorescence, and binding affinity for PD-L1 was expressed in normal organs. showed off-target binding to PD-L1, suggesting that it is clearly due to autoimmune activation (FIG. 20, bottom panel). However, Gluc25-S-aPD-L1 failed to release aPD-L1 in non-target tissues due to the lack of reducing environment and failed to target PD-L1 in non-target tissues. This may be the reason why Gluc25-S-aPD-L1 did not induce immune activation in non-target tissues. Based on the above, adverse events in non-target tissues can be avoided by performing ICB treatment using the antibody of the present invention that can specifically reactivate brain tissue, and clinical results are improved while suppressing side effects. It was suggested that
　上記実施例においては脳への抗体の送達を効率化するためにＰＥＧに脳の標的化分子としてＧＬＵＴ１リガンドを連結させた。以下では、脳以外への抗体送達とがんの治療実験のために、標的化分子を有しないＰＥＧ修飾抗体を作製した。図２１－１に示されるように、以下で用いる抗体は、非電荷親水性ポリマーブロックと還元環境応答性リンカーと抗体とがこの順番で連結した修飾抗体である。非電荷親水性ポリマーブロックとしては、上記同様にＰＥＧを用いた。ＰＥＧ化により抗体は抗原に対する結合活性を喪失した。 In the above example, the GLUT1 ligand was linked to PEG as a brain-targeting molecule in order to efficiently deliver antibodies to the brain. Below, PEG-modified antibodies without targeting molecules were generated for extrabrain antibody delivery and cancer therapy experiments. As shown in FIG. 21-1, the antibody used below is a modified antibody in which an uncharged hydrophilic polymer block, a reducing environment-responsive linker and an antibody are linked in that order. PEG was used as the non-charged hydrophilic polymer block in the same manner as described above. PEGylation caused the antibody to lose antigen-binding activity.
　悪性黒色腫細胞株の皮下移植モデルマウス（Ｂ１６－Ｆ１０）に対して、生理食塩水、未修飾の抗ＰＤ－Ｌ１抗体、および標的化分子を有しないＰＥＧ修飾抗ＰＤ－Ｌ１抗体を尾静注した（各実験群においてｎ＝５）。ＰＥＧ修飾抗ＰＤ－Ｌ１抗体は、上記実施例で作製した抗体と同様に作製した。投与量は、抗体量が１．５ｍｇ／ｋｇ体重となるように調節した。投与は、１日おきに３回行った。投与初日と３日おきに腫瘍体積（Ｖ）を測定し、腫瘍体積（Ｖ）の初日の腫瘍体積（Ｖ０）に対する比率（Ｖ／Ｖ０）を求めた。 Tail vein injection of physiological saline, unmodified anti-PD-L1 antibody, and PEG-modified anti-PD-L1 antibody without targeting molecule to subcutaneous transplantation mouse model of malignant melanoma cell line (B16-F10) (n=5 in each experimental group). A PEG-modified anti-PD-L1 antibody was produced in the same manner as the antibody produced in the above example. The dose was adjusted so that the amount of antibody was 1.5 mg/kg body weight. Dosing was performed three times every other day. The tumor volume (V) was measured on the first day of administration and every three days, and the ratio (V/V0) of the tumor volume (V) to the tumor volume (V0) on the first day was determined.
　結果は、図２１－２に示される通りであった。図２１－２に示されるように、生理食塩水投与群（Ｓａｌｉｎｅ）に対して、未修飾の抗ＰＤ－Ｌ１抗体投与群（Ｎａｔｉｖｅ　ａＰＤ－Ｌ１）は、有意な腫瘍体積増加の抑制作用を示した。これに対して、本発明の還元環境応答性のＰＥＧ修飾抗体投与群（ＰＥＧｙｌａｔｅｄ　ａＰＤ－Ｌ１）では、未修飾の抗ＰＤ－Ｌ１抗体投与群と比較しても、腫瘍体積の増加に対してさらに強い抑制作用を示した（図２１－２の左パネル）。次に、抗ＰＤ－Ｌ１抗体の代わりに抗ＰＤ－１抗体を用いる以外はすべて同じ条件で同様の実験を実施した。結果は、図２１－２の中央パネルに示される通りであった。抗体として抗ＰＤ－１抗体を用いた場合においても、効果は有意であり、本発明の還元環境応答性のＰＥＧ修飾抗体投与群（ＰＥＧｙｌａｔｅｄ　ａＰＤ－１）では、未修飾の抗ＰＤ－１抗体投与群と比較しても、腫瘍体積の増加に対してさらに強い抑制作用を示した（図２１－２の中央パネル）。この傾向は、抗体として抗ＣＴＬＡ－４抗体を用いた場合においても同様であった（図２１－２の右パネル）。 The results were as shown in Figure 21-2. As shown in FIG. 21-2, the unmodified anti-PD-L1 antibody administration group (Native aPD-L1) showed a significant suppressive effect on tumor volume increase compared to the saline administration group (Saline). rice field. In contrast, in the reducing environment-responsive PEG-modified antibody administration group (PEGylated aPD-L1) of the present invention, compared with the unmodified anti-PD-L1 antibody administration group, the tumor volume increased further. It showed a strong inhibitory action (left panel of FIG. 21-2). Next, a similar experiment was performed under the same conditions except that anti-PD-1 antibody was used instead of anti-PD-L1 antibody. The results were as shown in the middle panel of Figure 21-2. Even when an anti-PD-1 antibody was used as an antibody, the effect was significant. Compared with the group, it showed a stronger inhibitory effect on the increase in tumor volume (middle panel of Figure 21-2). This tendency was the same when an anti-CTLA-4 antibody was used as the antibody (right panel of FIG. 21-2).
　腫瘍組織内は、一般的に還元環境である。腫瘍は、細胞増殖に対して栄養や酸素供給が不足するために一般的に低酸素環境となる。低酸素環境下においてもがん細胞内に豊富に存在する還元酵素により腫瘍組織内は還元環境となる。本実施例では、ＰＥＧ修飾抗体について、還元環境応答性にＰＥＧを切断し、ネイティブの抗体に戻るように設計された。この抗体は、インビボにおいて現に腫瘍内の還元環境に応答して、喪失した抗原への結合活性を回復すること、および、未修飾抗体と同様の効果を発揮することが明らかとなった。ＰＥＧ修飾により、生体内の腫瘍以外への結合性は消失していると考えられ、したがって、腫瘍組織外での抗体による副作用は低減されていると合理的に考えられるが、その一方で、本発明のＰＥＧ修飾抗体は、未修飾抗体と同等以上の抗腫瘍効果を示した。このことは、修飾により腫瘍に送達される抗体量が増加したこと、すなわちＥＰＲ効果が増強されたことを示唆する。かくして、本発明のＰＥＧ修飾抗体は、修飾によりその結合活性を消失させることにより、その腫瘍組織外での副作用を低減すること、および／または腫瘍組織への送達量を増加させることができ、これによって、従来の未修飾抗体よりも低減された副作用および／または腫瘍組織への改善された送達量を達成し得る。 The inside of the tumor tissue is generally a reducing environment. Tumors generally have a hypoxic environment due to lack of nutrients and oxygen supply for cell growth. Even in a hypoxic environment, tumor tissue becomes a reductive environment due to reductase abundantly present in cancer cells. In this example, a PEG-modified antibody was designed to cleave the PEG and revert to a native antibody responsive to a reducing environment. This antibody was found to restore the lost antigen-binding activity in response to the reducing environment within the tumor in vivo, and to exert similar effects to the unmodified antibody. PEG modification is thought to eliminate in vivo binding properties other than tumors, and therefore, it is reasonably thought that the side effects of antibodies outside tumor tissues are reduced, but on the other hand, this The PEG-modified antibody of the invention showed an anti-tumor effect equal to or greater than that of the unmodified antibody. This suggests that the modification increased the amount of antibody delivered to the tumor, thus enhancing the EPR effect. Thus, the PEG-modified antibody of the present invention can reduce side effects outside of tumor tissue and/or increase the amount delivered to tumor tissue by eliminating its binding activity through modification. may achieve reduced side effects and/or improved delivery to tumor tissue than conventional unmodified antibodies.
抗体薬物コンジュゲート（ＡＤＣ）に対する本発明の修飾の適用
　抗体薬物コンジュゲートに対しても、本発明の修飾を適用することができる。ＡＤＣとしては、カドサイラ（ロッシュ社）を用いた。カドサイラは、トラスツズマブとエムタンシンがリンカーを介して連結して得られる下記式（Ｉ）記載のＡＤＣ（以下「Ｔ－ＤＭ１」ともいう）であり、抗がん剤として用いられている。 Application of the Modifications of the Invention to Antibody Drug Conjugates (ADCs) The modifications of the invention can also be applied to antibody drug conjugates. Kadcyla (Roche) was used as an ADC. Kadcyla is an ADC represented by the following formula (I) obtained by linking trastuzumab and emtansine via a linker (hereinafter also referred to as "T-DM1"), and is used as an anticancer agent.
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
　上記ＡＤＣ（未修飾型ＡＤＣ）に対して、ＰＥＧ修飾を施して修飾型ＡＤＣを得た。具体的には、ＰＥＧ修飾は、下記式（ＩＩ）に示されるＲ－ＰＥＧ－（ＣＨ２）２－ＳＳ－（ＣＨ２）２－ニトロフェニルカーボネート（ＮＰＣ）の構造を有する開裂性のリンカー－ＰＥＧを用いて行った。本実施例では、Ｒはメトキシ基とした。Ｒは、グルコースまたは反応性基（例えば、アジド基）などとすることもできる。反応性基には、体内の組織への標的化のための標的化分子を導入し得る。 The above ADC (unmodified ADC) was modified with PEG to obtain a modified ADC. Specifically, the PEG modification is a cleavable linker- PEG was used. In this example, R was a methoxy group. R can also be glucose or a reactive group such as an azide group. Reactive groups can incorporate targeting molecules for targeting to tissues within the body.
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
　本リンカーは、抗体に大量に存在するアミノ基のいくつかに連結し、還元環境下では、リンカーから開裂し、修飾ＡＤＣからＡＤＣ（未修飾型ＡＤＣ）を放出する。本試験では、ＡＤＣのＰＥＧ修飾および還元環境下でのＡＤＣ（未修飾型ＡＤＣ）の放出を試験した。 This linker is linked to some amino groups abundantly present in antibodies, and under a reducing environment, it is cleaved from the linker to release the ADC (unmodified ADC) from the modified ADC. In this study, PEG modification of ADC and release of ADC (unmodified ADC) under reducing environment were tested.
　カドサイラ　４．１０　ｍｇ／ｍｌ　（Ｔ－ＤＭ１　１．０　ｍｇ／ｍｌ）と２０ｍＭリン酸緩衝液（ｐＨ　７．４）中に溶かしたＰＥＧ－（ＣＨ２）２－ＳＳ－（ＣＨ２）２－ＮＰＣ　１０　ｍｇ／ｍｌを１対１（体積比）で混合し、室温で終夜反応させた。反応液を限外濾過（Ｖｉｖａｓｐｉｎ　６，　ＭＷＣＯ：　３０ｋＤａ，　４０００　ｇ，　４℃）で５回精製し、０．１　μｍ　ＰＶＤＦフィルターで処理してＰＥＧ修飾Ｔ－ＤＭ１を得た。粒径分布および散乱光強度をＺｅｔａｓｉｚｅｒ　Ｎａｎｏ　ＺＳ（５３２　ｎｍ，　１７３°）で測定した。対照としてＰＥＧ修飾していないＴ－ＤＭ１（未修飾型Ｔ－ＤＭ１）を測定した。結果は、図２２に示される通りであった。図２２に示されるように、ＰＥＧ修飾を受けたＴ－ＤＭ１（ＰＥＧ修飾Ｔ－ＤＭ１）は、未修飾型Ｔ－ＤＭ１と比較して平均流体力学径（体積平均）が明らかに増大していた。このことは、Ｔ－ＤＭ１がＰＥＧ修飾を受けたことを示唆する。 Kadcyla 4.10 mg/ml (T-DM1 1.0 mg/ml) and PEG-(CH 2 ) 2 -SS-(CH 2 ) 2 - dissolved in 20 mM phosphate buffer (pH 7.4) NPC 10 mg/ml was mixed 1:1 (volume ratio) and allowed to react overnight at room temperature. The reaction solution was purified by ultrafiltration (Vivaspin 6, MWCO: 30 kDa, 4000 g, 4° C.) five times and processed with a 0.1 μm PVDF filter to obtain PEG-modified T-DM1. Particle size distribution and scattered light intensity were measured with a Zetasizer Nano ZS (532 nm, 173°). As a control, T-DM1 not modified with PEG (unmodified T-DM1) was measured. The results were as shown in FIG. As shown in FIG. 22, PEG-modified T-DM1 (PEG-modified T-DM1) had a clearly increased average hydrodynamic diameter (volume average) compared to unmodified T-DM1. . This suggests that T-DM1 was PEG-modified.
　がん組織中では、グルタチオン（ＧＳＨ）濃度が高濃度であることが知られている。本試験では、がん組織中の還元環境を模擬するために１ｍＭのＧＳＨ存在下でＰＥＧ修飾Ｔ－ＤＭ１をインキュベートした（各ｎ＝３）。０、１、または３時間後に、粒径分布と散乱光強度を上記の通り測定した。対照として未修飾型Ｔ－ＤＭ１を測定した。結果は図２３に示される通りであった。図２３の左パネルに示されるように、ＰＥＧ修飾Ｔ－ＤＭ１は、ＧＳＨ存在下でのインキュベート時間が長くなるにつれて流体力学径（体積平均）を減少させた。また、図２３の右パネルに示されるように、ＰＥＧ修飾Ｔ－ＤＭ１の散乱光強度は、ＧＳＨ存在下での３時間のインキュベート後にＴ－ＤＭ１とほぼ同レベルまで低下した。このようにＰＥＧ修飾Ｔ－ＤＭ１は、がん組織中の還元環境下において開裂する。その結果、ＰＥＧ修飾Ｔ－ＤＭ１は、インタクトな未修飾型Ｔ－ＤＭ１を放出することが示唆された。 It is known that cancer tissue has a high concentration of glutathione (GSH). In this study, PEG-modified T-DM1 was incubated in the presence of 1 mM GSH to mimic the reducing environment in cancer tissue (n=3 each). After 0, 1, or 3 hours, the particle size distribution and scattered light intensity were measured as described above. Unmodified T-DM1 was measured as a control. The results were as shown in FIG. As shown in the left panel of FIG. 23, PEG-modified T-DM1 decreased hydrodynamic diameter (volume average) with increasing incubation time in the presence of GSH. Also, as shown in the right panel of FIG. 23, the scattered light intensity of PEG-modified T-DM1 decreased to approximately the same level as T-DM1 after 3 hours of incubation in the presence of GSH. Thus, PEG-modified T-DM1 is cleaved under the reducing environment in cancer tissue. The results suggested that PEG-modified T-DM1 released intact, unmodified T-DM1.
議論
　上記のように、本明細書では、標的分子未修飾型ＰＥＧ化プロセスおよびグルコース修飾型ＰＥＧ化プロセスに基づく再活性化可能なＩＣＢ抗体デリバリーシステムが開発された。適切に構成されたグルコース分子を持つＰＥＧ修飾抗体は、ＧＬＵＴ１を認識し、ＢＢＢの優先的かつ効率的な通過を促進することに成功した。また、標的分子未修飾型のＰＥＧ修飾抗体においても、抗体は未修飾抗体と同等以上の抗腫瘍効果を示した。このような送達システムの最も魅力的な利点は、標的組織または非標的組織での抗体機能のＯＮ－ＯＦＦ切り替えが可能であることであり、結合親和性がブロックされたＰＥＧ修飾抗体は、腫瘍内および脳内の還元的な環境を感知してＰＥＧ鎖の剥離を誘導し、その抗原に対する本来の結合性を回復させ、それによって強力に活性化された抗腫瘍免疫応答を脳組織特異的に引き出すことができる。これにより、脳組織外の腫瘍や原発性神経膠芽腫に対して抗腫瘍効果を発揮し、かつ、記憶免疫の誘導によって、長期的な腫瘍再発を防ぐことができる。対照的に、非標的組織（肺、肝臓、腎臓など）で完全な構造を維持したＰＥＧ修飾抗体は、その抗原特異性がブロックされており、免疫関連性の有害事象の発生を誘導しないように設計されている。これまでのところ、未修飾の免疫チェックポイント阻害抗体を用いた臨床でのＩＣＢ治療中に、薬物関連の有害事象は３９％の患者で観察され、グレード３または４の事象は９％の患者で指摘されている39。このように、今回の活性化可能なＩＣＢ療法の戦略は、将来的にｉｒＡＥの発生率を低下させ、神経膠芽腫に対して有益な結果をもたらす可能性のある臨床応用を促進するものである。 Discussion As noted above, reactivatable ICB antibody delivery systems based on target molecule-unmodified and glucose-modified PEGylation processes were developed herein. A PEG-modified antibody with appropriately configured glucose molecules successfully recognized GLUT1 and promoted preferential and efficient passage across the BBB. In addition, the target molecule-unmodified PEG-modified antibody also exhibited an anti-tumor effect equal to or greater than that of the unmodified antibody. The most attractive advantage of such delivery systems is the ability to switch antibody function ON-OFF in target or non-target tissues, and PEG-modified antibodies with blocked binding affinity can be used intratumorally. and sense the reductive environment in the brain to induce the shedding of PEG chains and restore their native binding to their antigens, thereby eliciting a potently activated anti-tumor immune response in a brain tissue-specific manner. be able to. As a result, it exerts an antitumor effect against tumors outside the brain tissue and primary glioblastoma, and can prevent long-term tumor recurrence by inducing memory immunity. In contrast, PEG-modified antibodies that retain their structural integrity in non-target tissues (e.g., lung, liver, kidney) have their antigen specificity blocked so as not to induce the development of immune-related adverse events. Designed. To date, drug-related adverse events have been observed in 39% of patients and grade 3 or 4 events in 9% of patients during clinical ICB treatment with unmodified immune checkpoint inhibitor antibodies. It has been pointed out39 . Thus, the present strategy of activatable ICB therapy will reduce the incidence of irAEs in the future, facilitating clinical applications with potentially beneficial outcomes in glioblastoma. be.
　ＰＥＧ化プロセスは、Ｄｏｘｉｌ23のようないくつかのＦＤＡ承認薬で証明されているように、ポリペプチド、他の候補分子またはナノキャリアにＰＥＧ鎖を連結させることは、迅速な消去、擬似アレルギー反応、および不十分な標的の蓄積などの医薬品の欠陥を克服するための長年の戦略である。このような活性化可能なＩＣＢ治療の有望なアプリケーションに鑑みれば、ＰＥＧ化抗体を用いた本発明の戦略は、合理的に設計されたタンパク質送達システムのための一般的かつ汎用性の高いプラットフォームとして利用することができる。例えば、酸性ｐＨ、酸化ストレス、ＴＭＥ内での過剰発現酵素などの反応特性を、適切な化学設計により部位特異的に活性化させることができる40。さらに、提案された標的部位に応じて適切なリガンドや抗体（生体高分子阻害剤やサイトカインも含む）を選択することが合理的である。したがって、ＰＥＧ化抗体の設計は、効率的なタンパク質送達のための大きな可能性を秘めており、生理的シグナルに基づいて様々な疾患を治療するための治療用タンパク質の設計へとさらに拡張される可能性がある。 The PEGylation process has been demonstrated in several FDA-approved drugs such as Doxil 23. Linking PEG chains to polypeptides, other candidate molecules or nanocarriers can lead to rapid elimination, pseudo-allergic reactions. , and poor target accumulation are long-standing strategies to overcome drug deficiencies. Given the promising application of such activatable ICB therapy, the present strategy using PEGylated antibodies could serve as a general and versatile platform for rationally designed protein delivery systems. can be used. For example, reactive properties such as acidic pH, oxidative stress, and overexpressed enzymes within the TME can be site-specifically activated by appropriate chemical design 40 . Furthermore, it is reasonable to select appropriate ligands and antibodies (including biopolymer inhibitors and cytokines) depending on the proposed target site. Therefore, the design of PEGylated antibodies has great potential for efficient protein delivery and is further extended to the design of therapeutic proteins to treat various diseases based on physiological signals. there is a possibility.
参考文献
17. Ye, Y., et al. Synergistic transcutaneous immunotherapy enhances antitumour immune responses through delivery of checkpoint inhibitors. ACS Nano 10, 8956-8963 (2016).
18. Chen, Q., et al. In situ sprayed bioresponsive immunotherapeutic gel for post-surgical cancer treatment. Nat. Nanotechnol. 14, 89-97 (2019).
19. Mi, Y., et al. A dual immunotherapy nanoparticle improves T-cell activation and cancer immunotherapy. Adv. Mater. 30, 1706098 (2018).
20. Bu, J., et al. An avidity-based PD-L1 antagonist using nanoparticle-antibody conjugates for enhanced immunotherapy. Nano Lett. 20, 4901-4909 (2020).
21. Hu, Q., et al. Conjugation of haematopoietic stem cells and platelets decorated with anti-PD-1 antibodies augments anti-leukaemia efficacy. Nat. Biomed. Eng. 2, 831-840 (2018).
22. Zhang, Y., et al. A tumour-targeted immune checkpoint blocker. Proc. Natl. Acad. Sci. 116, 15889-15894 (2019).
23. Harris, J.M. & Chess, R.B. Effect of pegylation on pharmaceuticals. Nat. Rev. Drug Discovery 2, 214-221 (2003).
24. Greenfield, N.J. Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 1, 2876-2890 (2006).
25. Louis-Jeune, C., Andrade-Navarro, M.A. & Perez-Iratxeta, C. Prediction of protein secondary structure from circular dichroism using theoretically derived spectra. Proteins 80, 374-381 (2012).
26. Wang, D., et al. Engineering nanoparticles to locally activate T cells in the tumour microenvironment. Sci. Immunol. 4, eaau6584 (2019).
27. Zhu, A., et al. Dually pH/reduction-responsive vesicles for ultrahigh-contrast fluorescence imaging and thermo-chemotherapy-synergized tumour ablation. ACS Nano 9, 7874-7885 (2015).
28. Wainwright, D.A., et al. Durable therapeutic efficacy utilizing combinatorial blockade against IDO, CTLA-4, and PD-L1 in mice with brain tumours. Clin. Cancer Res. 20, 5290-5301 (2014).
29. Thorens, B. & Mueckler, M. Glucose transporters in the 21st Century. Am. J. Physiol. 298, E141-E145 (2010).
30. Suzuki, K., et al. Glucose transporter 1-mediated vascular translocation of nanomedicines enhances accumulation and efficacy in solid tumours. J. Controlled Release 301, 28-41 (2019).
31. Anraku, Y., et al. Glycaemic control boosts glucosylated nanocarrier crossing the BBB into the brain. Nat. Commun. 8, 1001 (2017).
32. Xie, J., et al. Dual-sensitive nanomicelles enhancing systemic delivery of therapeutically active antibodies specifically into the brain. ACS Nano 14, 6729-6742 (2020).
33. Min, H.S., et al. Systemic brain delivery of antisense oligonucleotides across the blood-brain barrier with a glucose-coated polymeric nanocarrier. Angew. Chem. Int. Ed. 59, 8173-8180 (2020).
34. Cheng, F. & Eng, C. PTEN mutations trigger resistance to immunotherapy. Trends. Mol. Med. 25, 461-463 (2019).
35. Bao, S., et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756-760 (2006).
36. Sun, X., et al. Two-photon imaging of glutathione levels in intact brain indicates enhanced redox buffering in developing neurons and cells at the cerebrospinal fluid and blood-brain interface. J. Biol. Chem. 281, 17420-17431 (2006).
37. Harrison, F.E. & May, J.M. Vitamin C function in the brain: vital role of the ascorbate transporter SVCT2. Free Radical Biol. Med. 46, 719-730 (2009).
38. Ma, Q., et al. Calming cytokine storm in pneumonia by targeted delivery of TPCA-1 using platelet-derived extracellular vesicles. Matter 3, 287-301 (2020).
39. Brahmer, J.R., et al. Safety and activity of Anti-PD-L1 antibody in patients with advanced cancer. N. Engl. J. Med. 366, 2455-2465 (2012).
40. Blum, A.P., et al. Stimuli-responsive nanomaterials for biomedical applications. J. Am. Chem. Soc. 137, 2140-2154 (2015). References
17. Ye, Y., et al. Synergistic transcutaneous immunotherapy enhances antitumour immune responses through delivery of checkpoint inhibitors. ACS Nano 10, 8956-8963 (2016).
18. Chen, Q., et al. In situ sprayed bioresponsive immunotherapeutic gel for post-surgical cancer treatment. Nat. Nanotechnol. 14, 89-97 (2019).
19. Mi, Y., et al. A dual immunotherapy nanoparticle improves T-cell activation and cancer immunotherapy. Adv. Mater. 30, 1706098 (2018).
20. Bu, J., et al. An avidity-based PD-L1 antagonist using nanoparticle-antibody conjugates for enhanced immunotherapy. Nano Lett. 20, 4901-4909 (2020).
21. Hu, Q., et al. Conjugation of haematopoietic stem cells and platelets decorated with anti-PD-1 antibodies augments anti-leukaemia efficacy. Nat. Biomed. Eng. 2, 831-840 (2018).
22. Zhang, Y., et al. A tumor-targeted immune checkpoint blocker. Proc. Natl. Acad.
23. Harris, JM & Chess, RB Effect of pegylation on pharmaceuticals. Nat. Rev. Drug Discovery 2, 214-221 (2003).
24. Greenfield, NJ Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 1, 2876-2890 (2006).
25. Louis-Jeune, C., Andrade-Navarro, MA & Perez-Iratxeta, C. Prediction of protein secondary structure from circular dichroism using theoretically derived spectra. Proteins 80, 374-381 (2012).
26. Wang, D., et al. Engineering nanoparticles to locally activate T cells in the tumor microenvironment. Sci. Immunol. 4, eaau6584 (2019).
27. Zhu, A., et al. Dually pH/reduction-responsive vesicles for ultrahigh-contrast fluorescence imaging and thermo-chemotherapy-synergized tumor ablation. ACS Nano 9, 7874-7885 (2015).
28. Wainwright, DA, et al. Durable therapeutic efficacy utilizing combinatorial blockade against IDO, CTLA-4, and PD-L1 in mice with brain tumors. Clin. Cancer Res. 20, 5290-5301 (2014).
29. Thorens, B. & Mueckler, M. Glucose transporters in the 21st Century. Am. J. Physiol. 298, E141-E145 (2010).
30. Suzuki, K., et al. Glucose transporter 1-mediated vascular translocation of nanomedicines enhances accumulation and efficacy in solid tumors. J. Controlled Release 301, 28-41 (2019).
31. Anraku, Y., et al. Glycaemic control boosts glucosylated nanocarrier crossing the BBB into the brain. Nat. Commun. 8, 1001 (2017).
32. Xie, J., et al. Dual-sensitive nanomicelles enhancing systemic delivery of therapeutically active antibodies specifically into the brain. ACS Nano 14, 6729-6742 (2020).
33. Min, HS, et al. Systemic brain delivery of antisense oligonucleotides across the blood-brain barrier with a glucose-coated polymeric nanocarrier. Angew. Chem. Int. Ed. 59, 8173-8180 (2020).
34. Cheng, F. & Eng, C. PTEN mutations trigger resistance to immunotherapy. Trends. Mol. Med. 25, 461-463 (2019).
35. Bao, S., et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756-760 (2006).
36. Sun, X., et al. Two-photon imaging of glutathione levels in intact brain indicates enhanced redox buffering in developing neurons and cells at the cerebrospinal fluid and blood-brain interface. J. Biol. Chem. 281, 17420-17431 (2006).
37. Harrison, FE & May, JM Vitamin C function in the brain: vital role of the ascorbate transporter SVCT2. Free Radical Biol. Med. 46, 719-730 (2009).
38. Ma, Q., et al. Calming cytokine storm in pneumonia by targeted delivery of TPCA-1 using platelet-derived extracellular vesicles. Matter 3, 287-301 (2020).
39. Brahmer, JR, et al. Safety and activity of Anti-PD-L1 antibody in patients with advanced cancer. N. Engl. J. Med. 366, 2455-2465 (2012).
40. Blum, AP, et al. Stimuli-responsive nanomaterials for biomedical applications. J. Am. Chem. Soc. 137, 2140-2154 (2015).

Claims (20)

  1. 　非電荷親水性ポリマーブロックにより修飾された修飾抗体であって、非電荷親水性ポリマーブロック、環境応答性結合、および抗体が、この順番で連結し、各連結は、スペーサーを介していてもよく、環境応答性結合は、還元環境下で開裂する結合である、修飾抗体。 A modified antibody modified with an uncharged hydrophilic polymer block, wherein the uncharged hydrophilic polymer block, the environment-responsive linkage, and the antibody are linked in that order, each linkage optionally via a spacer, A modified antibody wherein the environment-responsive bond is a bond that cleaves under a reducing environment.
  2. 　その抗原に対する結合親和性（ＫＤ）が、前記修飾前と比較して、１０％以下である、請求項１に記載の抗体。 The antibody according to claim 1, wherein the binding affinity (KD) for the antigen is 10% or less compared to before the modification.
  3. 　その抗原に対する結合親和性（ＫＤ）が、前記修飾前と比較して、５％以下である、請求項１に記載の抗体。 The antibody according to claim 1, wherein the binding affinity (KD) for the antigen is 5% or less compared to before the modification.
  4. 　血清環境下において、その抗原に対して実質的にまたは有意に結合しない、請求項１に記載の抗体。 The antibody according to claim 1, which does not substantially or significantly bind to its antigen in a serum environment.
  5. 　環境応答性結合は、脳実質における還元環境下、または腫瘍組織における還元環境下において、開裂する結合である、請求項１～４のいずれか一項に記載の抗体。 The antibody according to any one of claims 1 to 4, wherein the environment-responsive bond is a bond that cleaves under a reducing environment in brain parenchyma or in a reducing environment in tumor tissue.
  6. 　環境応答性結合が開裂すると、その抗原に対する結合親和性（ＫＤ）が、回復する、請求項５に記載の抗体。 The antibody of claim 5, wherein the binding affinity (KD) for its antigen is restored upon cleavage of the environmentally responsive bond.
  7. 　－Ｃ（Ｏ）－Ｏ－Ｌ1－Ｓ－Ｓ－Ｌ2－（非電荷親水性ポリマーブロック）により修飾されたアミノ基を有する、請求項１～６のいずれか一項に記載の抗体
    ｛ここで、
    Ｌ1は置換されていてもよい低級アルキレンであり、
    Ｌ2は結合または生体内において安定なリンカーである｝。     The antibody according to any one of claims 1 to 6, which has an amino group modified by -C(O)-OL 1 -SSL 2 - (uncharged hydrophilic polymer block) { here,
    L 1 is optionally substituted lower alkylene,
    L2 is a bond or an in vivo stable linker } .
  8. 　Ｌ1が、エチレンである、請求項７に記載の抗体。 8. The antibody of claim 7 , wherein L1 is ethylene.
  9. 　ＰＤ－１系免疫チェックポイントを阻害する、請求項１～８のいずれか一項に記載の抗体。 The antibody according to any one of claims 1 to 8, which inhibits the PD-1 system immune checkpoint.
  10. 　ＰＤ－Ｌ１に結合する抗体である、請求項９に記載の抗体。 The antibody according to claim 9, which is an antibody that binds to PD-L1.
  11. 　がん抗原に結合する抗体である、請求項１～８のいずれか一項に記載の抗体。 The antibody according to any one of claims 1 to 8, which is an antibody that binds to a cancer antigen.
  12. 　標的化分子を表出している、請求項１～１１のいずれか一項に記載の抗体。 The antibody according to any one of claims 1 to 11, which expresses a targeting molecule.
  13. 　標的化分子が、非電荷親水性ポリマーブロックと連結している、請求項１２に記載の抗体。 The antibody of claim 12, wherein the targeting molecule is linked to an uncharged hydrophilic polymer block.
  14. 　標的化分子が、ＧＬＵＴ１リガンドである、請求項１３に記載の抗体。 The antibody according to claim 13, wherein the targeting molecule is a GLUT1 ligand.
  15. 　ＧＬＵＴ１リガンドが、グルコースである、請求項１４に記載の抗体。 The antibody of claim 14, wherein the GLUT1 ligand is glucose.
  16. 　免疫チェックポイント分子に結合して免疫チェックポイントを阻害する抗体は、免疫チェックポイント分子に結合して、免疫チェックポイント分子間の相互作用を中和する抗体である、請求項１～１５のいずれか一項に記載の抗体。 The antibody that binds to an immune checkpoint molecule and inhibits an immune checkpoint is an antibody that binds to an immune checkpoint molecule and neutralizes interaction between immune checkpoint molecules. The antibody according to item 1.
  17. 　免疫チェックポイント分子が、免疫細胞に発現する免疫チェックポイント分子のカウンターパートである、請求項１６に記載の抗体。 The antibody according to claim 16, wherein the immune checkpoint molecule is a counterpart of an immune checkpoint molecule expressed on immune cells.
  18. 　免疫チェックポイント分子が、免疫細胞に発現する免疫チェックポイント分子である、請求項１６に記載の抗体。 The antibody according to claim 16, wherein the immune checkpoint molecule is an immune checkpoint molecule expressed in immune cells.
  19. 　抗体が、抗体－薬物コンジュゲート（ＡＤＣ）の形態である、請求項１～１８のいずれか一項に記載の抗体。 The antibody according to any one of claims 1 to 18, wherein the antibody is in the form of an antibody-drug conjugate (ADC).
  20. 　請求項１～１９のいずれか一項に記載の抗体を含む、医薬組成物。


          A pharmaceutical composition comprising an antibody according to any one of claims 1-19.
PCT/JP2022/019622 2021-05-10 2022-05-09 Antibody having reduced binding affinity for antigen WO2022239720A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2023521007A JPWO2022239720A1 (en) 2021-05-10 2022-05-09
EP22807422.5A EP4342497A1 (en) 2021-05-10 2022-05-09 Antibody having reduced binding affinity for antigen

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021079655 2021-05-10
JP2021-079655 2021-05-10

Publications (1)

Publication Number Publication Date
WO2022239720A1 true WO2022239720A1 (en) 2022-11-17

Family

ID=84029624

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/019622 WO2022239720A1 (en) 2021-05-10 2022-05-09 Antibody having reduced binding affinity for antigen

Country Status (3)

Country Link
EP (1) EP4342497A1 (en)
JP (1) JPWO2022239720A1 (en)
WO (1) WO2022239720A1 (en)

Citations (169)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5443953A (en) 1993-12-08 1995-08-22 Immunomedics, Inc. Preparation and use of immunoconjugates
US5525338A (en) 1992-08-21 1996-06-11 Immunomedics, Inc. Detection and therapy of lesions with biotin/avidin conjugates
US5587459A (en) 1994-08-19 1996-12-24 Regents Of The University Of Minnesota Immunoconjugates comprising tyrosine kinase inhibitors
US5677136A (en) 1994-11-14 1997-10-14 Systemix, Inc. Methods of obtaining compositions enriched for hematopoietic stem cells, compositions derived therefrom and methods of use thereof
US5686072A (en) 1992-06-17 1997-11-11 Board Of Regents, The University Of Texas Epitope-specific monoclonal antibodies and immunotoxins and uses thereof
US5716595A (en) 1992-05-06 1998-02-10 Immunomedics, Inc. Intraperative, intravascular and endoscopic tumor and lesion detection and therapy with monovalent antibody fragments
US5736119A (en) 1993-05-17 1998-04-07 Immunomedics, Inc. Detection and therapy of lesions with biotin/avidin-metal chelating protein conjugates
US5776456A (en) 1992-11-13 1998-07-07 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US5789554A (en) 1994-08-12 1998-08-04 Immunomedics, Inc. Immunoconjugates and humanized antibodies specific for B-cell lymphoma and leukemia cells
US5798229A (en) 1993-08-02 1998-08-25 Merck Patent Gesellschaft Mit Beschrankter Haftung Bispecific molecules recognizing lymphocyte antigen CD2 and tumor antigens
US5814440A (en) 1995-06-07 1998-09-29 Systemix, Inc. Methods of obtaining compositions enriched for hematopoietic stem cells, antibodies for use therein, compositions derived therefrom and methods of use thereof
US5874540A (en) 1994-10-05 1999-02-23 Immunomedics, Inc. CDR-grafted type III anti-CEA humanized mouse monoclonal antibodies
US6077499A (en) 1996-05-03 2000-06-20 Immunomedics, Inc. Targeted combination immunotherapy of cancer
US6096289A (en) 1992-05-06 2000-08-01 Immunomedics, Inc. Intraoperative, intravascular, and endoscopic tumor and lesion detection, biopsy and therapy
US6107090A (en) 1996-05-06 2000-08-22 Cornell Research Foundation, Inc. Treatment and diagnosis of prostate cancer with antibodies to extracellur PSMA domains
US6120767A (en) 1986-10-27 2000-09-19 Pharmaceutical Royalties, L.L.C. Chimeric antibody with specificity to human B cell surface antigen
US6129914A (en) 1992-03-27 2000-10-10 Protein Design Labs, Inc. Bispecific antibody effective to treat B-cell lymphoma and cell line
US6183744B1 (en) 1997-03-24 2001-02-06 Immunomedics, Inc. Immunotherapy of B-cell malignancies using anti-CD22 antibodies
US6254868B1 (en) 1996-03-20 2001-07-03 Immunomedics, Inc. Glycosylated humanized B-cell specific antibodies
US6306393B1 (en) 1997-03-24 2001-10-23 Immunomedics, Inc. Immunotherapy of B-cell malignancies using anti-CD22 antibodies
US6331175B1 (en) 1985-07-05 2001-12-18 Immunomedics, Inc. Method and kit for imaging and treating organs and tissues
US6340459B1 (en) 1995-12-01 2002-01-22 The Trustees Of Columbia University In The City Of New York Therapeutic applications for the anti-T-BAM (CD40-L) monoclonal antibody 5C8 in the treatment of reperfusion injury in non-transplant recipients
US6340571B1 (en) 1996-03-21 2002-01-22 Bio Merieux Antibodies specific for Staphylococcus aureus, and use thereof
US6344198B1 (en) 1995-01-20 2002-02-05 Human Genome Sciences, Inc. Human prostatic specific reductase
US6346246B1 (en) 1994-01-25 2002-02-12 Human Genome Sciences, Inc. Antibodies that bind human haemopoietic maturation factor
US6355444B1 (en) 1992-01-29 2002-03-12 Viventia Biotech Inc. Carcinoma associated antigen (SK1) monoclonal antibodies against SK1, methods of producing these antibodies and uses therefor
US6355245B1 (en) 1994-05-02 2002-03-12 Alexion Pharmaceuticals, Inc. C5-specific antibodies for the treatment of inflammatory diseases
US6355481B1 (en) 1999-06-18 2002-03-12 Emory University Hybridoma cell line and monoclonal antibody for huntingtin protein
US6355244B1 (en) 1997-11-17 2002-03-12 University Of Kentucky Research Foundation Methods and compositions for the treatment of psoriasis
US6359126B1 (en) 1992-04-03 2002-03-19 Genentech, Inc. Antibodies to αvβ3 integrin
US6372215B1 (en) 1997-03-03 2002-04-16 Bristol-Myers Squibb Company Monoclonal antibodies to human CD6
US6376654B1 (en) 1999-08-13 2002-04-23 Molecular Discoveries, Llc Myeloma cell and ovarian cancer cell surface glycoproteins, antibodies thereto, and uses thereof
US6383759B1 (en) 1998-05-04 2002-05-07 Gerald P. Murphy Cancer Foundation Non-invasive method to detect prostate cancer
US6383484B1 (en) 1998-12-21 2002-05-07 Ludwig Institute For Cancer Research Antibodies to truncated VEGF-D and thereof
US6395276B1 (en) 1997-05-02 2002-05-28 Immunomedics, Inc. Immunotoxins directed against malignant cells
US6395274B1 (en) 1993-11-19 2002-05-28 Baylor College Of Medicine Monoclonal antibodies specific for human interleukin-5
US6403770B1 (en) 1996-10-25 2002-06-11 Human Genome Sciences, Inc. Antibodies to neutrokine-alpha
US6403091B1 (en) 1991-11-15 2002-06-11 The Trustees Of Columbia University In The City Of New York Methods for inhibiting the rejection of a transplant organ in a subject with 5C8-specific antibodies
US6406694B1 (en) 1998-07-23 2002-06-18 Millennium Pharmaceuticals, Inc. Anti-CCR2 antibodies
US6413726B1 (en) 1996-03-19 2002-07-02 Human Genome Sciences, Inc. Antibodies that specifically bind Cytostatin III
US6419928B1 (en) 1988-12-22 2002-07-16 Genzyme Corporation Monoclonal antibodies to transforming growth factor-beta and methods of use
US6432404B1 (en) 1993-12-23 2002-08-13 Icos Corporation Methods of inhibiting locomotor damage following spinal cord injury with α D-specific antibodies
US6432402B1 (en) 1989-05-25 2002-08-13 Sloan-Kettering Institute For Cancer Research Anti-idiotypic antibody which induces an immune response against a glycosphingolipid and use thereof
US6441143B1 (en) 1995-04-13 2002-08-27 Amgen Inc Methods and compositions for determining HER-2/neu expression
US6444206B1 (en) 1993-05-28 2002-09-03 The Scripps Research Institute Methods and compositions for inhibiting CD14 mediated cell activation
US6455040B1 (en) 1997-01-14 2002-09-24 Human Genome Sciences, Inc. Tumor necrosis factor receptor 5
US6458356B1 (en) 1995-12-05 2002-10-01 Amgen Inc. Antibody-induced apoptosis
US6461823B1 (en) 1997-01-28 2002-10-08 Human Genome Sciences, Inc. Death domain containing receptor-4 antibodies
US6465173B2 (en) 1992-03-09 2002-10-15 Immune Research, Ltd. Anti-idiotypic antibody and its use in diagnosis and therapy in HIV-related disease
US6468531B1 (en) 1993-09-09 2002-10-22 Duke University Method of promoting cellular function
US6468529B1 (en) 1995-06-02 2002-10-22 Genentech, Inc. Hepatocyte growth factor receptor antagonists and uses thereof
US6479247B1 (en) 1996-10-09 2002-11-12 The Corporation Of The Trustees Of The Order Of The Sisters Of Mercy In Queensland Dendritic cell-specific antibodies
US6482598B2 (en) 1996-11-13 2002-11-19 Morphogenesis, Inc. Antibody recognizing a small subset of human hematopoietic cells
US6482408B2 (en) 1995-06-05 2002-11-19 Human Genome Sciences, Inc. Fibroblast growth factor 15 antibodies
US6488930B1 (en) 1999-01-15 2002-12-03 Millennium Pharmaceuticals, Inc. Anti-CCR4 antibodies and methods of use therefor
US6511665B1 (en) 1987-11-25 2003-01-28 Immunex Corporation Antibodies to interleukin-1 receptors
US6518404B1 (en) 1994-10-17 2003-02-11 Human Genome Sciences, Inc. Human endothelin-bombesin receptor antibodies
US6521227B1 (en) 1999-11-18 2003-02-18 Peter L. Hudson Polynucleotides encoding prostatic growth factor and process for producing prostatic growth factor polypeptides
US6528625B1 (en) 1996-10-28 2003-03-04 Millennium Pharmaceuticals, Inc. Anti-CCR5 antibodies and kits comprising same
US6528269B1 (en) 1998-06-22 2003-03-04 Case Western Reserve University Immunological agents specific for prion protein (PRP)
US6534058B2 (en) 2000-10-10 2003-03-18 Tanox, Inc. Anti-C5 monoclonal antibodies
US6544749B1 (en) 1992-05-08 2003-04-08 Genentech, Inc. Antibodies to leukemia inhibitory factor and their use in immunoassays
US6545130B2 (en) 1997-06-04 2003-04-08 Albert Einstein College Of Medicine Of Yeshiva University Monoclonal antibodies to mycobacterium tuberculosis and a modified ELISA assay
US6562618B1 (en) 1997-12-25 2003-05-13 Japan Tobacco, Inc. Monoclonal antibody against connective tissue growth factor and medicinal uses thereof
US6566076B1 (en) 1998-04-23 2003-05-20 The Regents Of The University Of California Detection and diagnosis of conditions associated with lung injury
US6572856B1 (en) 1998-09-10 2003-06-03 The University Of Virginia Patent Foundation Methods for the prevention and treatment of cancer using anti-C3b(i) antibodies
US6576745B1 (en) 1996-04-03 2003-06-10 Human Genome Sciences, Inc. Human cystatin F antibodies
US6596852B2 (en) 1994-07-08 2003-07-22 Immunex Corporation Antibodies that bind the cytokine designated LERK-5
US6605441B1 (en) 1995-06-05 2003-08-12 Human Genome Sciences, Inc. Antibodies against fibroblast growth factor 11
US6605279B2 (en) 1993-07-26 2003-08-12 Genetics Institute, Inc. Therapeutic compositions for inhibiting the interactions of B7-1 and B7-2 with their natural ligands
US6610833B1 (en) 1997-11-24 2003-08-26 The Institute For Human Genetics And Biochemistry Monoclonal human natural antibodies
US6630144B1 (en) 1999-08-30 2003-10-07 The United States Of America As Represented By The Secretary Of The Army Monoclonal antibodies to Ebola glycoprotein
US6653104B2 (en) 1996-10-17 2003-11-25 Immunomedics, Inc. Immunotoxins, comprising an internalizing antibody, directed against malignant and normal cells
US6652852B1 (en) 1986-10-27 2003-11-25 Royalty Pharma Finance Trust Chimeric antibody with specificity to human B cell surface antigen
US6673344B1 (en) 1994-08-23 2004-01-06 Human Genome Sciences, Inc. Antibodies to human CKβ-10/MCP-4
US6682736B1 (en) 1998-12-23 2004-01-27 Abgenix, Inc. Human monoclonal antibodies to CTLA-4
US6682737B1 (en) 1996-03-29 2004-01-27 North Carolina State University Anti-cryptosporidium parvum preparations
US6689355B2 (en) 2000-05-11 2004-02-10 Altarex Corp. Therapeutic method and composition utilizing antigen-antibody complexation and presentation by dendritic cells
US6689607B2 (en) 1997-10-21 2004-02-10 Human Genome Sciences, Inc. Human tumor, necrosis factor receptor-like proteins TR11, TR11SV1 and TR11SV2
US6689362B1 (en) 1997-06-03 2004-02-10 Regents Of The University Of Minnesota Method for treating T-lineage leukemias and lymphomas using a CD7-specific monoclonal antibody (TXU-7) linked to the pokeweed antiviral protein (PAP)
US6692908B1 (en) 1998-11-05 2004-02-17 Stanford University Prevention and treatment of HCV infection employing antibodies that inhibit the interaction of HCV virions with their receptor
US6693176B1 (en) 1999-07-23 2004-02-17 University Of Massachusetts Antitumor antibodies, proteins, and uses thereof
US6709653B1 (en) 1994-09-16 2004-03-23 Human Genome Sciences, Inc. Antibodies specific for human inositol monophosphatase H1
US6716966B1 (en) 1999-08-18 2004-04-06 Altarex Corp. Therapeutic binding agents against MUC-1 antigen and methods for their use
US6720155B1 (en) 1998-08-13 2004-04-13 Angel Lopez Monoclonal antibody inhibitor of GM-CSF, IL-3, IL-5 and other cytokines, and uses thereof
US6730300B2 (en) 1996-03-20 2004-05-04 Immunomedics, Inc. Humanization of an anti-carcinoembryonic antigen anti-idiotype antibody and use as a tumor vaccine and for targeting applications
US6733981B2 (en) 1994-11-01 2004-05-11 Human Genome Sciences, Inc. Antibodies to interleukin-1 β converting enzyme like apoptosis protease-3 and 4
US6743898B2 (en) 2001-03-15 2004-06-01 Ochsner Clinic Foundation Monoclonal antibodies that suppress B cell growth and/or differentiation
US6764681B2 (en) 1991-10-07 2004-07-20 Biogen, Inc. Method of prophylaxis or treatment of antigen presenting cell driven skin conditions using inhibitors of the CD2/LFA-3 interaction
US6764688B2 (en) 1996-09-03 2004-07-20 Kaneka Corporation Method for inducing immunosuppressive cells and a culture device to be used therefor
US6764679B2 (en) 1997-09-18 2004-07-20 Genentech, Inc. Antibodies to DcR3 Polypeptide, a TNFR Homolog
US6767711B2 (en) 1995-06-05 2004-07-27 Cornell Research Foundation, Inc. Treatment and diagnosis of prostate cancer
US6770450B1 (en) 1996-05-06 2004-08-03 Cornell Research Foundation, Inc. Treatment and diagnosis of cancer
US6783758B2 (en) 1999-11-08 2004-08-31 Rhode Island Hospital Diagnosis and treatment of malignant neoplasms
US6824778B2 (en) 2001-04-23 2004-11-30 The United States Of America As Represented By The Secretary Of The Army Prophylactic and therapeutic monoclonal antibodies
US6824780B1 (en) 1999-10-29 2004-11-30 Genentech, Inc. Anti-tumor antibody compositions and methods of use
US6835549B2 (en) 2000-02-24 2004-12-28 University Of Medicine & Dentistry Of New Jersey Immunoassay method for the diagnosis of gastric intestinal metaplasia associated with gastric carcinoma
US6835370B2 (en) 1999-11-08 2004-12-28 Rhode Island Hospital Diagnosis and treatment of malignant neoplasms
US6861226B2 (en) 1996-03-21 2005-03-01 Human Genome Sciences, Inc. Human endometrial specific steroid-binding factor I, II and III
US6861511B1 (en) 1986-06-04 2005-03-01 Bayer Corporation Detection and quantification of neu related proteins in the biological fluids of humans
US6861227B2 (en) 1998-03-19 2005-03-01 Human Genome Sciences, Inc. Antibodies to cytokine receptor common gamma chain like
US6864062B2 (en) 1999-06-16 2005-03-08 Molecular Geriatrics Corporation Purified antigen for Alzheimer's disease and methods of obtaining and using same
US6867006B2 (en) 1994-05-16 2005-03-15 Human Genome Sciences, Inc. Antibodies to human chemotactic protein
US6872568B1 (en) 1997-03-17 2005-03-29 Human Genome Sciences, Inc. Death domain containing receptor 5 antibodies
US6875580B2 (en) 2003-01-28 2005-04-05 Schering Corporation Antibodies specific for plasmacytoid dendritic cells
US6878812B2 (en) 1997-01-21 2005-04-12 Human Genome Science, Inc. Metalloproteinases
US6881405B2 (en) 1998-06-15 2005-04-19 Altarex Medical Corp. Reagents and methods for inducing an immune response to prostate specific antigen
US6884594B2 (en) 1996-03-26 2005-04-26 Human Genome Sciences, Inc. Antibodies to growth factor HTTER36
US6887468B1 (en) 1999-04-28 2005-05-03 Board Of Regents, The University Of Texas System Antibody kits for selectively inhibiting VEGF
US6887466B2 (en) 1988-11-23 2005-05-03 Genetics Institute, Inc. Methods for selectively stimulating proliferation of T cells
US6899879B2 (en) 1992-07-09 2005-05-31 Chiron Corporation Method for treating an IgE-mediated disease in a patient using anti-CD40 monoclonal antibodies
US6899864B2 (en) 2001-03-30 2005-05-31 Immunomedics, Inc. Morpholino imaging and therapy
US6919433B2 (en) 1997-03-14 2005-07-19 Human Genome Sciences, Inc. Antibodies to protein HPMBQ91
US6919078B2 (en) 1997-06-11 2005-07-19 Human Genome Sciences, Inc. Antibodies to human tumor necrosis factor receptor TR9
US6921533B2 (en) 1989-06-02 2005-07-26 The Johns Hopkins University School Of Medicine Method of using monoclonal antibodies against leukocyte adhesion receptor β-chain
US6921645B2 (en) 1994-08-23 2005-07-26 Human Genome Sciences, Inc. Antibodies to chemokine β-4
US6926893B1 (en) 1994-07-06 2005-08-09 Immunomedics, Inc. Multi-stage cascade boosting vaccine
US6939547B2 (en) 2000-07-31 2005-09-06 The United States Of America As Represented By The Department Of Health And Human Services Specific binding agents for KSHV vIL-6 that neutralize a biological activity
US6946129B1 (en) 1999-06-08 2005-09-20 Seattle Genetics, Inc. Recombinant anti-CD40 antibody and uses thereof
US6949244B1 (en) 1995-12-20 2005-09-27 The Board Of Trustees Of The University Of Kentucky Murine monoclonal anti-idiotype antibody 11D10 and methods of use thereof
US6951924B2 (en) 1997-03-14 2005-10-04 Human Genome Sciences, Inc. Antibodies against secreted protein HTEBYII
US6956107B2 (en) 1998-02-20 2005-10-18 Tanox, Inc. Inhibitors of complement activation
US6962702B2 (en) 1998-06-22 2005-11-08 Immunomedics Inc. Production and use of novel peptide-based agents for use with bi-specific antibodies
US6962981B1 (en) 1996-03-25 2005-11-08 Medarex, Inc. Monoclonal antibodies specific for the extracellular domain of prostate-specific membrane antigen
US6962813B2 (en) 2001-05-21 2005-11-08 The Brigham And Women's Hospital, Inc. P. aeruginosa mucoid exopolysaccharide specific binding peptides
US6965018B2 (en) 2000-06-06 2005-11-15 Bristol-Myers Squibb Company Antibodies directed to B7-related polypeptide, BSL-2
US6964854B1 (en) 1999-07-13 2005-11-15 Science & Technology Corporation Compositions and methods useful for the diagnosis and treatment of heparin induced thrombocytopenia/thrombosis
US20050271671A1 (en) 2003-01-24 2005-12-08 Immunomedics, Inc. Anthracycline-antibody conjugates
US6974863B2 (en) 1988-11-07 2005-12-13 Indiana University Research And Technology Corporation Antibody for 4-1BB
US6989241B2 (en) 2000-10-02 2006-01-24 Oklahoma Medical Research Foundation Assay for rapid detection of human activated protein C and highly specific monoclonal antibody therefor
US6994852B1 (en) 1999-11-12 2006-02-07 Temple University-Of The Commonwealth System Of Higher Education Inhibition of angiogenesis by antibodies against high molecular weight kininogen domain 5
US6994976B1 (en) 1999-11-19 2006-02-07 Tittle Thomas V Tr3-specific binding agents and methods for their use
US6998468B2 (en) 2000-03-23 2006-02-14 Tanox, Inc. Anti-C2/C2a inhibitors of complement activation
US7001598B2 (en) 1995-01-26 2006-02-21 Biogen Idec Ma Inc. Anti-lymphotoxin-beta receptor antibodies as anti-tumor agents
US7012133B1 (en) 1995-01-19 2006-03-14 Children's Medical Center Corp. Antibodies to C-C chemokine Receptor 3 Protein
US7037498B2 (en) 2001-01-05 2006-05-02 Abgenix, Inc. Antibodies to insulin-like growth factor I receptor
US7038018B2 (en) 1999-11-24 2006-05-02 Millennium Pharmaceuticals, Inc. Antibodies and ligands for “Bonzo” chemokine receptor
US7041802B2 (en) 1998-12-23 2006-05-09 Human Genome Sciences, Inc. Peptidoglycan recognition proteins
US7041803B2 (en) 1997-01-21 2006-05-09 Human Genome Sciences, Inc. Galectin 11
US7041293B1 (en) 1990-04-03 2006-05-09 Genentech, Inc. HIV env antibodies
US7045132B2 (en) 1991-11-14 2006-05-16 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, C/O Centers For Disease Control And Prevention Streptococcus pneumoniae 37-kDa surface adhesin a protein
US7049060B2 (en) 2001-11-05 2006-05-23 Ortho-Clinical Diagnostics, Inc. HCV anti-core monoclonal antibodies
US7060802B1 (en) 2000-09-18 2006-06-13 The Trustees Of Columbia University In The City Of New York Tumor-associated marker
US7074403B1 (en) 1999-06-09 2006-07-11 Immunomedics, Inc. Immunotherapy of autoimmune disorders using antibodies which target B-cells
US20060193865A1 (en) 2002-12-13 2006-08-31 Immunomedics, Inc. Camptothecin-binding moiety conjugates
US7109304B2 (en) 2003-07-31 2006-09-19 Immunomedics, Inc. Humanized anti-CD19 antibodies
US20060210475A1 (en) 2005-03-03 2006-09-21 Goldenberg David M Humanized L243 antibodies
US7151164B2 (en) 2002-02-14 2006-12-19 Immunomedics, Inc. Anti-CD20 antibodies and fusion proteins thereof and methods of use
US20070087001A1 (en) 2005-10-19 2007-04-19 Center For Molecular Medicine And Immunology Inhibition of placenta growth factor (PLGF) mediated metastasis and/or angiogenesis
US7230084B2 (en) 1998-05-20 2007-06-12 Immunomedics, Inc. Therapeutic using a bispecific antibody
US7238785B2 (en) 2002-03-01 2007-07-03 Immunomedics, Inc. RS7 antibodies
US7238786B2 (en) 2002-06-14 2007-07-03 Immunomedics, Inc. Monoclonal antibody cPAM4
US7251164B2 (en) 2004-11-10 2007-07-31 Innovative Silicon S.A. Circuitry for and method of improving statistical distribution of integrated circuits
US7256004B2 (en) 1998-10-31 2007-08-14 United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Variants of humanized anti-carcinoma monoclonal antibody CC49
US7282567B2 (en) 2002-06-14 2007-10-16 Immunomedics, Inc. Monoclonal antibody hPAM4
US7300655B2 (en) 2002-08-01 2007-11-27 Immunomedics, Inc. Alpha-fetoprotein Immu31 antibodies and fusion proteins and methods of use thereof
US7312318B2 (en) 2002-03-01 2007-12-25 Immunomedics, Inc. Internalizing anti-CD74 antibodies and methods of use
US7387773B2 (en) 2001-08-07 2008-06-17 Massey University Vaccine
US7531327B2 (en) 2004-07-23 2009-05-12 Immunomedics, Inc. Methods and compositions for increasing longevity and protein yield from a cell culture
US7537930B2 (en) 2004-07-23 2009-05-26 Immunomedics, Inc. Mammalian cell lines for increasing longevity and protein yield from a cell culture
US7541440B2 (en) 2002-09-30 2009-06-02 Immunomedics, Inc. Chimeric, human and humanized anti-granulocyte antibodies and methods of use
US7585491B2 (en) 2002-12-13 2009-09-08 Immunomedics, Inc. Immunoconjugates with an intracellularly-cleavable linkage
US7608425B2 (en) 2004-07-23 2009-10-27 Immunomedics, Inc. Methods for protein expression in mammalian cells in serum-free medium
WO2009130575A2 (en) 2008-04-22 2009-10-29 Universita' Degli Studi Di Verona Isolated monoclonal antibody or fragment thereof binding prostate specific membrane antigen, conjugates and uses thereof
WO2010083536A1 (en) * 2009-01-19 2010-07-22 Bayer Healthcare Llc Protein conjugate having an endopeptidase-cleavable bioprotective moiety
WO2012112689A1 (en) 2011-02-15 2012-08-23 The University Of North Carolina At Chapel Hill Nanoparticle, liposomes, polymers, agents and proteins modified with reversible linkers
WO2015075942A1 (en) 2013-11-22 2015-05-28 国立大学法人 東京大学 Carrier for drug delivery and conjugate, composition containing same, and method for administering same
WO2017002979A1 (en) * 2015-07-02 2017-01-05 国立大学法人 東京大学 Drug delivery carrier, and composition containing same
WO2021230375A1 (en) * 2020-05-15 2021-11-18 公益財団法人川崎市産業振興財団 Peptide modified with glut1-ligand-modified non-charged hydrophilic polymer cleavably under reductive environment or low-ph environment, and antibody comprising said peptide

Patent Citations (192)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6331175B1 (en) 1985-07-05 2001-12-18 Immunomedics, Inc. Method and kit for imaging and treating organs and tissues
US6861511B1 (en) 1986-06-04 2005-03-01 Bayer Corporation Detection and quantification of neu related proteins in the biological fluids of humans
US6120767A (en) 1986-10-27 2000-09-19 Pharmaceutical Royalties, L.L.C. Chimeric antibody with specificity to human B cell surface antigen
US6893625B1 (en) 1986-10-27 2005-05-17 Royalty Pharma Finance Trust Chimeric antibody with specificity to human B cell surface antigen
US6652852B1 (en) 1986-10-27 2003-11-25 Royalty Pharma Finance Trust Chimeric antibody with specificity to human B cell surface antigen
US6511665B1 (en) 1987-11-25 2003-01-28 Immunex Corporation Antibodies to interleukin-1 receptors
US6974863B2 (en) 1988-11-07 2005-12-13 Indiana University Research And Technology Corporation Antibody for 4-1BB
US6887466B2 (en) 1988-11-23 2005-05-03 Genetics Institute, Inc. Methods for selectively stimulating proliferation of T cells
US6419928B1 (en) 1988-12-22 2002-07-16 Genzyme Corporation Monoclonal antibodies to transforming growth factor-beta and methods of use
US6432402B1 (en) 1989-05-25 2002-08-13 Sloan-Kettering Institute For Cancer Research Anti-idiotypic antibody which induces an immune response against a glycosphingolipid and use thereof
US6921533B2 (en) 1989-06-02 2005-07-26 The Johns Hopkins University School Of Medicine Method of using monoclonal antibodies against leukocyte adhesion receptor β-chain
US7041293B1 (en) 1990-04-03 2006-05-09 Genentech, Inc. HIV env antibodies
US6764681B2 (en) 1991-10-07 2004-07-20 Biogen, Inc. Method of prophylaxis or treatment of antigen presenting cell driven skin conditions using inhibitors of the CD2/LFA-3 interaction
US7045132B2 (en) 1991-11-14 2006-05-16 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, C/O Centers For Disease Control And Prevention Streptococcus pneumoniae 37-kDa surface adhesin a protein
US6455044B1 (en) 1991-11-15 2002-09-24 The Trustees Of Columbia University In The City Of New York 5c8 antigen
US6403091B1 (en) 1991-11-15 2002-06-11 The Trustees Of Columbia University In The City Of New York Methods for inhibiting the rejection of a transplant organ in a subject with 5C8-specific antibodies
US6451310B1 (en) 1991-11-15 2002-09-17 The Trustees Of Columbia University In The City Of New York Method for inhibiting an allergic response with a 5c8-specific antibody
US6592868B1 (en) 1991-11-15 2003-07-15 The Trustees Of Columbia University In The City Of New York Methods for treating autoimmune diseases with 5C8-specific antibodies
US6610294B1 (en) 1991-11-15 2003-08-26 The Trustees Of Columbia University In The City Of New York Methods of inhibiting an autoimmune response in a human suffering from an autoimmune disease by administering an antibody that binds to a protein to which monoclonal antibody 5C8 binds
US6793924B2 (en) 1991-11-15 2004-09-21 The Trustees Of Columbia University In The City Of New York Protein recognized by an antibody that specifically binds an epitope that is specifically bound by monoclonal antibody 5c8.
US6355444B1 (en) 1992-01-29 2002-03-12 Viventia Biotech Inc. Carcinoma associated antigen (SK1) monoclonal antibodies against SK1, methods of producing these antibodies and uses therefor
US6916475B2 (en) 1992-03-09 2005-07-12 Hema Diagnostic Systems L.L.C. Anti-idiotypic antibody and its use in regulating the composition of T cell lymphocytes
US6465173B2 (en) 1992-03-09 2002-10-15 Immune Research, Ltd. Anti-idiotypic antibody and its use in diagnosis and therapy in HIV-related disease
US6129914A (en) 1992-03-27 2000-10-10 Protein Design Labs, Inc. Bispecific antibody effective to treat B-cell lymphoma and cell line
US6359126B1 (en) 1992-04-03 2002-03-19 Genentech, Inc. Antibodies to αvβ3 integrin
US6096289A (en) 1992-05-06 2000-08-01 Immunomedics, Inc. Intraoperative, intravascular, and endoscopic tumor and lesion detection, biopsy and therapy
US5716595A (en) 1992-05-06 1998-02-10 Immunomedics, Inc. Intraperative, intravascular and endoscopic tumor and lesion detection and therapy with monovalent antibody fragments
US6387350B2 (en) 1992-05-06 2002-05-14 Immunomedics, Inc. Intraoperative, intravascular and endoscopic tumor and lesion detection, biopsy and therapy
US6544749B1 (en) 1992-05-08 2003-04-08 Genentech, Inc. Antibodies to leukemia inhibitory factor and their use in immunoassays
US5686072A (en) 1992-06-17 1997-11-11 Board Of Regents, The University Of Texas Epitope-specific monoclonal antibodies and immunotoxins and uses thereof
US6899879B2 (en) 1992-07-09 2005-05-31 Chiron Corporation Method for treating an IgE-mediated disease in a patient using anti-CD40 monoclonal antibodies
US5525338A (en) 1992-08-21 1996-06-11 Immunomedics, Inc. Detection and therapy of lesions with biotin/avidin conjugates
US5776456A (en) 1992-11-13 1998-07-07 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US6682734B1 (en) 1992-11-13 2004-01-27 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US5922302A (en) 1993-05-17 1999-07-13 Immunomedics, Inc. Detection and therapy of lesions with biotin/avidin-metal chelating protein conjugates
US5736119A (en) 1993-05-17 1998-04-07 Immunomedics, Inc. Detection and therapy of lesions with biotin/avidin-metal chelating protein conjugates
US6444206B1 (en) 1993-05-28 2002-09-03 The Scripps Research Institute Methods and compositions for inhibiting CD14 mediated cell activation
US6605279B2 (en) 1993-07-26 2003-08-12 Genetics Institute, Inc. Therapeutic compositions for inhibiting the interactions of B7-1 and B7-2 with their natural ligands
US5798229A (en) 1993-08-02 1998-08-25 Merck Patent Gesellschaft Mit Beschrankter Haftung Bispecific molecules recognizing lymphocyte antigen CD2 and tumor antigens
US6468531B1 (en) 1993-09-09 2002-10-22 Duke University Method of promoting cellular function
US6395274B1 (en) 1993-11-19 2002-05-28 Baylor College Of Medicine Monoclonal antibodies specific for human interleukin-5
US5443953A (en) 1993-12-08 1995-08-22 Immunomedics, Inc. Preparation and use of immunoconjugates
US6432404B1 (en) 1993-12-23 2002-08-13 Icos Corporation Methods of inhibiting locomotor damage following spinal cord injury with α D-specific antibodies
US6346246B1 (en) 1994-01-25 2002-02-12 Human Genome Sciences, Inc. Antibodies that bind human haemopoietic maturation factor
US6355245B1 (en) 1994-05-02 2002-03-12 Alexion Pharmaceuticals, Inc. C5-specific antibodies for the treatment of inflammatory diseases
US6867006B2 (en) 1994-05-16 2005-03-15 Human Genome Sciences, Inc. Antibodies to human chemotactic protein
US6905681B1 (en) 1994-06-03 2005-06-14 Genetics Institute, Inc. Methods for selectively stimulating proliferation of T cells
US6926893B1 (en) 1994-07-06 2005-08-09 Immunomedics, Inc. Multi-stage cascade boosting vaccine
US6596852B2 (en) 1994-07-08 2003-07-22 Immunex Corporation Antibodies that bind the cytokine designated LERK-5
US5789554A (en) 1994-08-12 1998-08-04 Immunomedics, Inc. Immunoconjugates and humanized antibodies specific for B-cell lymphoma and leukemia cells
US6187287B1 (en) 1994-08-12 2001-02-13 Immunomedics, Inc. Immunoconjugates and humanized antibodies specific for B-cell lymphoma and leukemia cells
US5587459A (en) 1994-08-19 1996-12-24 Regents Of The University Of Minnesota Immunoconjugates comprising tyrosine kinase inhibitors
US6673344B1 (en) 1994-08-23 2004-01-06 Human Genome Sciences, Inc. Antibodies to human CKβ-10/MCP-4
US6921645B2 (en) 1994-08-23 2005-07-26 Human Genome Sciences, Inc. Antibodies to chemokine β-4
US6709653B1 (en) 1994-09-16 2004-03-23 Human Genome Sciences, Inc. Antibodies specific for human inositol monophosphatase H1
US5874540A (en) 1994-10-05 1999-02-23 Immunomedics, Inc. CDR-grafted type III anti-CEA humanized mouse monoclonal antibodies
US6676924B2 (en) 1994-10-05 2004-01-13 Immunomedics, Inc. CDR-grafted type III anti-CEA humanized mouse monoclonal antibodies
US6518404B1 (en) 1994-10-17 2003-02-11 Human Genome Sciences, Inc. Human endothelin-bombesin receptor antibodies
US6733981B2 (en) 1994-11-01 2004-05-11 Human Genome Sciences, Inc. Antibodies to interleukin-1 β converting enzyme like apoptosis protease-3 and 4
US5677136A (en) 1994-11-14 1997-10-14 Systemix, Inc. Methods of obtaining compositions enriched for hematopoietic stem cells, compositions derived therefrom and methods of use thereof
US7012133B1 (en) 1995-01-19 2006-03-14 Children's Medical Center Corp. Antibodies to C-C chemokine Receptor 3 Protein
US6344198B1 (en) 1995-01-20 2002-02-05 Human Genome Sciences, Inc. Human prostatic specific reductase
US7001598B2 (en) 1995-01-26 2006-02-21 Biogen Idec Ma Inc. Anti-lymphotoxin-beta receptor antibodies as anti-tumor agents
US6441143B1 (en) 1995-04-13 2002-08-27 Amgen Inc Methods and compositions for determining HER-2/neu expression
US6468529B1 (en) 1995-06-02 2002-10-22 Genentech, Inc. Hepatocyte growth factor receptor antagonists and uses thereof
US6482408B2 (en) 1995-06-05 2002-11-19 Human Genome Sciences, Inc. Fibroblast growth factor 15 antibodies
US6767711B2 (en) 1995-06-05 2004-07-27 Cornell Research Foundation, Inc. Treatment and diagnosis of prostate cancer
US6605441B1 (en) 1995-06-05 2003-08-12 Human Genome Sciences, Inc. Antibodies against fibroblast growth factor 11
US5814440A (en) 1995-06-07 1998-09-29 Systemix, Inc. Methods of obtaining compositions enriched for hematopoietic stem cells, antibodies for use therein, compositions derived therefrom and methods of use thereof
US6340459B1 (en) 1995-12-01 2002-01-22 The Trustees Of Columbia University In The City Of New York Therapeutic applications for the anti-T-BAM (CD40-L) monoclonal antibody 5C8 in the treatment of reperfusion injury in non-transplant recipients
US6458356B1 (en) 1995-12-05 2002-10-01 Amgen Inc. Antibody-induced apoptosis
US6949244B1 (en) 1995-12-20 2005-09-27 The Board Of Trustees Of The University Of Kentucky Murine monoclonal anti-idiotype antibody 11D10 and methods of use thereof
US6413726B1 (en) 1996-03-19 2002-07-02 Human Genome Sciences, Inc. Antibodies that specifically bind Cytostatin III
US6730300B2 (en) 1996-03-20 2004-05-04 Immunomedics, Inc. Humanization of an anti-carcinoembryonic antigen anti-idiotype antibody and use as a tumor vaccine and for targeting applications
US6254868B1 (en) 1996-03-20 2001-07-03 Immunomedics, Inc. Glycosylated humanized B-cell specific antibodies
US6340571B1 (en) 1996-03-21 2002-01-22 Bio Merieux Antibodies specific for Staphylococcus aureus, and use thereof
US6861226B2 (en) 1996-03-21 2005-03-01 Human Genome Sciences, Inc. Human endometrial specific steroid-binding factor I, II and III
US6962981B1 (en) 1996-03-25 2005-11-08 Medarex, Inc. Monoclonal antibodies specific for the extracellular domain of prostate-specific membrane antigen
US6884594B2 (en) 1996-03-26 2005-04-26 Human Genome Sciences, Inc. Antibodies to growth factor HTTER36
US6730307B2 (en) 1996-03-29 2004-05-04 The Arizona Board Of Regents Acting On Behalf Of The University Of Arizona Anti-cryptosporidium parvum preparations
US6682737B1 (en) 1996-03-29 2004-01-27 North Carolina State University Anti-cryptosporidium parvum preparations
US6576745B1 (en) 1996-04-03 2003-06-10 Human Genome Sciences, Inc. Human cystatin F antibodies
US6077499A (en) 1996-05-03 2000-06-20 Immunomedics, Inc. Targeted combination immunotherapy of cancer
US6770450B1 (en) 1996-05-06 2004-08-03 Cornell Research Foundation, Inc. Treatment and diagnosis of cancer
US6107090A (en) 1996-05-06 2000-08-22 Cornell Research Foundation, Inc. Treatment and diagnosis of prostate cancer with antibodies to extracellur PSMA domains
US6764688B2 (en) 1996-09-03 2004-07-20 Kaneka Corporation Method for inducing immunosuppressive cells and a culture device to be used therefor
US6479247B1 (en) 1996-10-09 2002-11-12 The Corporation Of The Trustees Of The Order Of The Sisters Of Mercy In Queensland Dendritic cell-specific antibodies
US6653104B2 (en) 1996-10-17 2003-11-25 Immunomedics, Inc. Immunotoxins, comprising an internalizing antibody, directed against malignant and normal cells
US6403770B1 (en) 1996-10-25 2002-06-11 Human Genome Sciences, Inc. Antibodies to neutrokine-alpha
US6635482B1 (en) 1996-10-25 2003-10-21 Human Genome Sciences, Inc. Monoclonal antibodies to membrane neutrokine-α
US6528625B1 (en) 1996-10-28 2003-03-04 Millennium Pharmaceuticals, Inc. Anti-CCR5 antibodies and kits comprising same
US6482598B2 (en) 1996-11-13 2002-11-19 Morphogenesis, Inc. Antibody recognizing a small subset of human hematopoietic cells
US6838282B2 (en) 1996-11-13 2005-01-04 Morphogenesis, Inc. Antibody recognizing a small subset of human hematopoietic cells
US6455040B1 (en) 1997-01-14 2002-09-24 Human Genome Sciences, Inc. Tumor necrosis factor receptor 5
US7041803B2 (en) 1997-01-21 2006-05-09 Human Genome Sciences, Inc. Galectin 11
US6878812B2 (en) 1997-01-21 2005-04-12 Human Genome Science, Inc. Metalloproteinases
US6943020B2 (en) 1997-01-28 2005-09-13 Human Genome Sciences, Inc. Death domain containing receptor-4 antibodies
US6461823B1 (en) 1997-01-28 2002-10-08 Human Genome Sciences, Inc. Death domain containing receptor-4 antibodies
US6372215B1 (en) 1997-03-03 2002-04-16 Bristol-Myers Squibb Company Monoclonal antibodies to human CD6
US6919433B2 (en) 1997-03-14 2005-07-19 Human Genome Sciences, Inc. Antibodies to protein HPMBQ91
US6951924B2 (en) 1997-03-14 2005-10-04 Human Genome Sciences, Inc. Antibodies against secreted protein HTEBYII
US6872568B1 (en) 1997-03-17 2005-03-29 Human Genome Sciences, Inc. Death domain containing receptor 5 antibodies
US6306393B1 (en) 1997-03-24 2001-10-23 Immunomedics, Inc. Immunotherapy of B-cell malignancies using anti-CD22 antibodies
US6183744B1 (en) 1997-03-24 2001-02-06 Immunomedics, Inc. Immunotherapy of B-cell malignancies using anti-CD22 antibodies
US6395276B1 (en) 1997-05-02 2002-05-28 Immunomedics, Inc. Immunotoxins directed against malignant cells
US6689362B1 (en) 1997-06-03 2004-02-10 Regents Of The University Of Minnesota Method for treating T-lineage leukemias and lymphomas using a CD7-specific monoclonal antibody (TXU-7) linked to the pokeweed antiviral protein (PAP)
US6545130B2 (en) 1997-06-04 2003-04-08 Albert Einstein College Of Medicine Of Yeshiva University Monoclonal antibodies to mycobacterium tuberculosis and a modified ELISA assay
US6919078B2 (en) 1997-06-11 2005-07-19 Human Genome Sciences, Inc. Antibodies to human tumor necrosis factor receptor TR9
US6764679B2 (en) 1997-09-18 2004-07-20 Genentech, Inc. Antibodies to DcR3 Polypeptide, a TNFR Homolog
US6689607B2 (en) 1997-10-21 2004-02-10 Human Genome Sciences, Inc. Human tumor, necrosis factor receptor-like proteins TR11, TR11SV1 and TR11SV2
US6355244B1 (en) 1997-11-17 2002-03-12 University Of Kentucky Research Foundation Methods and compositions for the treatment of psoriasis
US6610833B1 (en) 1997-11-24 2003-08-26 The Institute For Human Genetics And Biochemistry Monoclonal human natural antibodies
US6562618B1 (en) 1997-12-25 2003-05-13 Japan Tobacco, Inc. Monoclonal antibody against connective tissue growth factor and medicinal uses thereof
US6956107B2 (en) 1998-02-20 2005-10-18 Tanox, Inc. Inhibitors of complement activation
US6861227B2 (en) 1998-03-19 2005-03-01 Human Genome Sciences, Inc. Antibodies to cytokine receptor common gamma chain like
US6566076B1 (en) 1998-04-23 2003-05-20 The Regents Of The University Of California Detection and diagnosis of conditions associated with lung injury
US6383759B1 (en) 1998-05-04 2002-05-07 Gerald P. Murphy Cancer Foundation Non-invasive method to detect prostate cancer
US7230084B2 (en) 1998-05-20 2007-06-12 Immunomedics, Inc. Therapeutic using a bispecific antibody
US6881405B2 (en) 1998-06-15 2005-04-19 Altarex Medical Corp. Reagents and methods for inducing an immune response to prostate specific antigen
US6962702B2 (en) 1998-06-22 2005-11-08 Immunomedics Inc. Production and use of novel peptide-based agents for use with bi-specific antibodies
US6528269B1 (en) 1998-06-22 2003-03-04 Case Western Reserve University Immunological agents specific for prion protein (PRP)
US6406694B1 (en) 1998-07-23 2002-06-18 Millennium Pharmaceuticals, Inc. Anti-CCR2 antibodies
US6491915B2 (en) 1998-07-23 2002-12-10 Millennium Pharmaceuticals, Inc. Anti-CCR2 antibodies and methods of use therefor
US6720155B1 (en) 1998-08-13 2004-04-13 Angel Lopez Monoclonal antibody inhibitor of GM-CSF, IL-3, IL-5 and other cytokines, and uses thereof
US6572856B1 (en) 1998-09-10 2003-06-03 The University Of Virginia Patent Foundation Methods for the prevention and treatment of cancer using anti-C3b(i) antibodies
US7256004B2 (en) 1998-10-31 2007-08-14 United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Variants of humanized anti-carcinoma monoclonal antibody CC49
US6692908B1 (en) 1998-11-05 2004-02-17 Stanford University Prevention and treatment of HCV infection employing antibodies that inhibit the interaction of HCV virions with their receptor
US6383484B1 (en) 1998-12-21 2002-05-07 Ludwig Institute For Cancer Research Antibodies to truncated VEGF-D and thereof
US6682736B1 (en) 1998-12-23 2004-01-27 Abgenix, Inc. Human monoclonal antibodies to CTLA-4
US7041802B2 (en) 1998-12-23 2006-05-09 Human Genome Sciences, Inc. Peptidoglycan recognition proteins
US6488930B1 (en) 1999-01-15 2002-12-03 Millennium Pharmaceuticals, Inc. Anti-CCR4 antibodies and methods of use therefor
US7056509B2 (en) 1999-04-28 2006-06-06 Board Of Regents The University Of Texas System Antibody methods for selectively inhibiting VEGF
US6887468B1 (en) 1999-04-28 2005-05-03 Board Of Regents, The University Of Texas System Antibody kits for selectively inhibiting VEGF
US6946129B1 (en) 1999-06-08 2005-09-20 Seattle Genetics, Inc. Recombinant anti-CD40 antibody and uses thereof
US7074403B1 (en) 1999-06-09 2006-07-11 Immunomedics, Inc. Immunotherapy of autoimmune disorders using antibodies which target B-cells
US6864062B2 (en) 1999-06-16 2005-03-08 Molecular Geriatrics Corporation Purified antigen for Alzheimer's disease and methods of obtaining and using same
US6355481B1 (en) 1999-06-18 2002-03-12 Emory University Hybridoma cell line and monoclonal antibody for huntingtin protein
US6964854B1 (en) 1999-07-13 2005-11-15 Science & Technology Corporation Compositions and methods useful for the diagnosis and treatment of heparin induced thrombocytopenia/thrombosis
US6693176B1 (en) 1999-07-23 2004-02-17 University Of Massachusetts Antitumor antibodies, proteins, and uses thereof
US6376654B1 (en) 1999-08-13 2002-04-23 Molecular Discoveries, Llc Myeloma cell and ovarian cancer cell surface glycoproteins, antibodies thereto, and uses thereof
US6716966B1 (en) 1999-08-18 2004-04-06 Altarex Corp. Therapeutic binding agents against MUC-1 antigen and methods for their use
US6630144B1 (en) 1999-08-30 2003-10-07 The United States Of America As Represented By The Secretary Of The Army Monoclonal antibodies to Ebola glycoprotein
US6824780B1 (en) 1999-10-29 2004-11-30 Genentech, Inc. Anti-tumor antibody compositions and methods of use
US6812206B2 (en) 1999-11-08 2004-11-02 Rhode Island Hospital Diagnosis and treatment of malignant neoplasms
US6783758B2 (en) 1999-11-08 2004-08-31 Rhode Island Hospital Diagnosis and treatment of malignant neoplasms
US6835370B2 (en) 1999-11-08 2004-12-28 Rhode Island Hospital Diagnosis and treatment of malignant neoplasms
US6994852B1 (en) 1999-11-12 2006-02-07 Temple University-Of The Commonwealth System Of Higher Education Inhibition of angiogenesis by antibodies against high molecular weight kininogen domain 5
US6521227B1 (en) 1999-11-18 2003-02-18 Peter L. Hudson Polynucleotides encoding prostatic growth factor and process for producing prostatic growth factor polypeptides
US6994976B1 (en) 1999-11-19 2006-02-07 Tittle Thomas V Tr3-specific binding agents and methods for their use
US7038018B2 (en) 1999-11-24 2006-05-02 Millennium Pharmaceuticals, Inc. Antibodies and ligands for “Bonzo” chemokine receptor
US6835549B2 (en) 2000-02-24 2004-12-28 University Of Medicine & Dentistry Of New Jersey Immunoassay method for the diagnosis of gastric intestinal metaplasia associated with gastric carcinoma
US6998468B2 (en) 2000-03-23 2006-02-14 Tanox, Inc. Anti-C2/C2a inhibitors of complement activation
US6689355B2 (en) 2000-05-11 2004-02-10 Altarex Corp. Therapeutic method and composition utilizing antigen-antibody complexation and presentation by dendritic cells
US6965018B2 (en) 2000-06-06 2005-11-15 Bristol-Myers Squibb Company Antibodies directed to B7-related polypeptide, BSL-2
US6939547B2 (en) 2000-07-31 2005-09-06 The United States Of America As Represented By The Department Of Health And Human Services Specific binding agents for KSHV vIL-6 that neutralize a biological activity
US7060802B1 (en) 2000-09-18 2006-06-13 The Trustees Of Columbia University In The City Of New York Tumor-associated marker
US6989241B2 (en) 2000-10-02 2006-01-24 Oklahoma Medical Research Foundation Assay for rapid detection of human activated protein C and highly specific monoclonal antibody therefor
US6534058B2 (en) 2000-10-10 2003-03-18 Tanox, Inc. Anti-C5 monoclonal antibodies
US7037498B2 (en) 2001-01-05 2006-05-02 Abgenix, Inc. Antibodies to insulin-like growth factor I receptor
US6743898B2 (en) 2001-03-15 2004-06-01 Ochsner Clinic Foundation Monoclonal antibodies that suppress B cell growth and/or differentiation
US6899864B2 (en) 2001-03-30 2005-05-31 Immunomedics, Inc. Morpholino imaging and therapy
US6824778B2 (en) 2001-04-23 2004-11-30 The United States Of America As Represented By The Secretary Of The Army Prophylactic and therapeutic monoclonal antibodies
US6962813B2 (en) 2001-05-21 2005-11-08 The Brigham And Women's Hospital, Inc. P. aeruginosa mucoid exopolysaccharide specific binding peptides
US7387773B2 (en) 2001-08-07 2008-06-17 Massey University Vaccine
US7049060B2 (en) 2001-11-05 2006-05-23 Ortho-Clinical Diagnostics, Inc. HCV anti-core monoclonal antibodies
US7151164B2 (en) 2002-02-14 2006-12-19 Immunomedics, Inc. Anti-CD20 antibodies and fusion proteins thereof and methods of use
US7312318B2 (en) 2002-03-01 2007-12-25 Immunomedics, Inc. Internalizing anti-CD74 antibodies and methods of use
US7238785B2 (en) 2002-03-01 2007-07-03 Immunomedics, Inc. RS7 antibodies
US7282567B2 (en) 2002-06-14 2007-10-16 Immunomedics, Inc. Monoclonal antibody hPAM4
US7238786B2 (en) 2002-06-14 2007-07-03 Immunomedics, Inc. Monoclonal antibody cPAM4
US7300655B2 (en) 2002-08-01 2007-11-27 Immunomedics, Inc. Alpha-fetoprotein Immu31 antibodies and fusion proteins and methods of use thereof
US7541440B2 (en) 2002-09-30 2009-06-02 Immunomedics, Inc. Chimeric, human and humanized anti-granulocyte antibodies and methods of use
US7585491B2 (en) 2002-12-13 2009-09-08 Immunomedics, Inc. Immunoconjugates with an intracellularly-cleavable linkage
US20060193865A1 (en) 2002-12-13 2006-08-31 Immunomedics, Inc. Camptothecin-binding moiety conjugates
US20050271671A1 (en) 2003-01-24 2005-12-08 Immunomedics, Inc. Anthracycline-antibody conjugates
US6875580B2 (en) 2003-01-28 2005-04-05 Schering Corporation Antibodies specific for plasmacytoid dendritic cells
US7109304B2 (en) 2003-07-31 2006-09-19 Immunomedics, Inc. Humanized anti-CD19 antibodies
US7608425B2 (en) 2004-07-23 2009-10-27 Immunomedics, Inc. Methods for protein expression in mammalian cells in serum-free medium
US7537930B2 (en) 2004-07-23 2009-05-26 Immunomedics, Inc. Mammalian cell lines for increasing longevity and protein yield from a cell culture
US7785880B2 (en) 2004-07-23 2010-08-31 Immunomedics, Inc. Mammalian cell lines for increasing longevity and protein yield from cell culture
US7531327B2 (en) 2004-07-23 2009-05-12 Immunomedics, Inc. Methods and compositions for increasing longevity and protein yield from a cell culture
US7251164B2 (en) 2004-11-10 2007-07-31 Innovative Silicon S.A. Circuitry for and method of improving statistical distribution of integrated circuits
US7612180B2 (en) 2005-03-03 2009-11-03 Immunomedics, Inc. Humanized L243 antibodies
US20060210475A1 (en) 2005-03-03 2006-09-21 Goldenberg David M Humanized L243 antibodies
US20070087001A1 (en) 2005-10-19 2007-04-19 Center For Molecular Medicine And Immunology Inhibition of placenta growth factor (PLGF) mediated metastasis and/or angiogenesis
US7642239B2 (en) 2005-10-19 2010-01-05 Center For Molecular Medicine And Immunology Inhibition of placenta growth factor (PLGF) mediated metastasis and/or angiogenesis
WO2009130575A2 (en) 2008-04-22 2009-10-29 Universita' Degli Studi Di Verona Isolated monoclonal antibody or fragment thereof binding prostate specific membrane antigen, conjugates and uses thereof
WO2010083536A1 (en) * 2009-01-19 2010-07-22 Bayer Healthcare Llc Protein conjugate having an endopeptidase-cleavable bioprotective moiety
WO2012112689A1 (en) 2011-02-15 2012-08-23 The University Of North Carolina At Chapel Hill Nanoparticle, liposomes, polymers, agents and proteins modified with reversible linkers
WO2015075942A1 (en) 2013-11-22 2015-05-28 国立大学法人 東京大学 Carrier for drug delivery and conjugate, composition containing same, and method for administering same
WO2017002979A1 (en) * 2015-07-02 2017-01-05 国立大学法人 東京大学 Drug delivery carrier, and composition containing same
WO2021230375A1 (en) * 2020-05-15 2021-11-18 公益財団法人川崎市産業振興財団 Peptide modified with glut1-ligand-modified non-charged hydrophilic polymer cleavably under reductive environment or low-ph environment, and antibody comprising said peptide

Non-Patent Citations (45)

* Cited by examiner, † Cited by third party
Title
ALDAPE, K. ET AL.: "Challenges to curing primary brain tumours", NAT. REV. CLIN. ONCOL., vol. 16, 2019, pages 509 - 520, XP037085252, DOI: 10.1038/s41571-019-0177-5
ANRAKU, Y. ET AL.: "Glycaemic control boosts glucosylated nanocarrier crossing the BBB into the brain", NAT. COMMUN, vol. 8, 2017, pages 1001, XP055533568, DOI: 10.1038/s41467-017-00952-3
ARVANITIS, C.D.FERRARO, G.BJAIN, R.K.: "The blood-brain barrier and blood-tumour barrier in brain tumours and metastases", NAT. REV. CANCER., vol. 20, 2020, pages 26 - 41, XP036974943, DOI: 10.1038/s41568-019-0205-x
BAO, S. ET AL.: "Glioma stem cells promote radioresistance by preferential activation of the DNA damage response", NATURE, vol. 444, 2006, pages 756 - 760, XP055200572, DOI: 10.1038/nature05236
BINNEWIES, M. ET AL.: "Understanding the tumour immune microenvironment (TIME) for effective therapy", NAT. MED., vol. 24, 2018, pages 541 - 550
BLUM, A.P. ET AL.: "Stimuli-responsive nanomaterials for biomedical applications", J. AM. CHEM. SOC., vol. 137, 2015, pages 2140 - 2154
BRAHMER, J.R. ET AL.: "Safety and activity of Anti-PD-L1 antibody in patients with advanced cancer", N. ENGL. J. MED., vol. 366, 2012, pages 2455 - 2465, XP002685330, DOI: 10.1056/NEJMoa1200694
BU, J. ET AL.: "An avidity-based PD-L1 antagonist using nanoparticle-antibody conjugates for enhanced immunotherapy", NANO LETT., vol. 20, 2020, pages 4901 - 4909
CHEN, Q. ET AL.: "In situ sprayed bioresponsive immunotherapeutic gel for post-surgical cancer treatment", NAT. NANOTECHNOL., vol. 14, 2019, pages 89 - 97, XP036667512, DOI: 10.1038/s41565-018-0319-4
CHENG, F.ENG, C: "PTEN mutations trigger resistance to immunotherapy", TRENDS. MOL. MED, vol. 25, 2019, pages 461 - 463
GAJEWSKI, T.F.SCHREIBER, HFU, Y.-X: "Innate and adaptive immune cells in the tumour microenvironment", NAT. IMMUNOL., vol. 14, 2013, pages 1014 - 1022
GREENFIELD, N.J.: "Using circular dichroism spectra to estimate protein secondary structure", NAT. PROTOC., vol. 1, 2006, pages 2876 - 2890, XP055220113, DOI: 10.1038/nprot.2006.202
HARRIS, J.M.CHESS, R.B: "Effect of pegylation on pharmaceuticals", NAT. REV. DRUG DISCOVERY, vol. 2, 2003, pages 214 - 221, XP009042217, DOI: 10.1038/nrd1033
HARRISON, F.EMAY, J.M: "Vitamin C function in the brain: vital role of the ascorbate transporter SVCT2", FREE RADICAL BIOL. MED, vol. 46, 2009, pages 719 - 730, XP055558047, DOI: 10.1016/j.freeradbiomed.2008.12.018
HERBST, R.S. ET AL.: "Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients", NATURE, vol. 515, 2014, pages 563 - 567, XP037922694, DOI: 10.1038/nature14011
HU, Q. ET AL.: "Conjugation of haematopoietic stem cells and platelets decorated with anti-PD-1 antibodies augments anti-leukaemia efficacy", NAT. BIOMED. ENG., vol. 2, 2018, pages 831 - 840, XP036629635, DOI: 10.1038/s41551-018-0310-2
JOSHUA D. THOMAS; TERRENCE R. BURKE;: "Application of a water-soluble pyridyl disulfide amine linker for use in Cu-free click bioconjugation", TETRAHEDRON LETTERS, ELSEVIER, AMSTERDAM , NL, vol. 52, no. 33, 9 June 2011 (2011-06-09), Amsterdam , NL , pages 4316 - 4319, XP028237756, ISSN: 0040-4039, DOI: 10.1016/j.tetlet.2011.06.042 *
KABAT, E.A. ET AL.: "Sequences of Proteins of Immunological Interest", 1991, US DEPT. OF HEALTH AND HUMAN SERVICES
LIM, M.XIA, Y.BETTEGOWDA, CWELLER, M: "Current state of immunotherapy for glioblastoma", NAT. REV. CLIN. ONCOL., vol. 15, 2018, pages 422 - 442, XP036529744, DOI: 10.1038/s41571-018-0003-5
LOUIS-JEUNE, C.ANDRADE-NAVARRO, M.APEREZ-IRATXETA, C: "Prediction of protein secondary structure from circular dichroism using theoretically derived spectra", PROTEINS, vol. 80, 2012, pages 374 - 381, XP055668205
MA, Q. ET AL.: "alming cytokine storm in pneumonia by targeted delivery of TPCA-1 using platelet-derived extracellular vesicles", MATTER, vol. 3, 2020, pages 287 - 301, XP055781110, DOI: 10.1016/j.matt.2020.05.017
MARTINS, F. ET AL.: "Adverse effects of immune-checkpoint inhibitors: epidemiology, management and surveillance", NAT. REV. CLIN. ONCOL., vol. 16, 2019, pages 563 - 580, XP036867466, DOI: 10.1038/s41571-019-0218-0
MCGRANAHAN, T.THERKELSEN, K.E.AHMAD, SNAGPAL, S: "Current state of immunotherapy for treatment of glioblastoma", CURR. TREAT. OPTIONS IN ONCOL, vol. 20, 2019, pages 24
MELLMAN, I.COUKOS, GDRANOFF, G: "Cancer immunotherapy comes of age", NATURE, vol. 480, 2011, pages 480 - 489, XP055054665, DOI: 10.1038/nature10673
MI, Y. ET AL.: "A dual immunotherapy nanoparticle improves T-cell activation and cancer immunotherapy", ADV. MATER, vol. 30, 2018, pages 1706098
MIN, H.S. ET AL.: "Systemic brain delivery of antisense oligonucleotides across the blood-brain barrier with a glucose-coated polymeric nanocarrier", ANGEW. CHEM. INT. ED., vol. 59, 2020, pages 8173 - 8180
NAIDOO, J. ET AL.: "Toxicities of the anti-PD-1 and anti-PD-L1 immune checkpoint antibodies", ANN. ONCOL, vol. 26, 2015, pages 2375 - 2391
PARDOLL, D.M.: "The blockade of immune checkpoints in cancer immunotherapy", NAT. REV. CANCER., vol. 12, 2012, pages 252 - 264, XP037114946, DOI: 10.1038/nrc3239
POSTOW, M.A.SIDLOW, R.HELLMANN, M.D: "Immune-related adverse events associated with immune checkpoint blockade", N. ENGL. J. MED., vol. 378, 2018, pages 158 - 168
RAMOS-CASALS, M. ET AL.: "Immune-related adverse events of checkpoint inhibitors", NAT. REV. DIS. PRIMERS, vol. 6, 2020, pages 38, XP037131257, DOI: 10.1038/s41572-020-0160-6
ROSENBERG, J.E. ET AL.: "Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial", THE LANCET, vol. 387, 2016, pages 1909 - 1920, XP029530539, DOI: 10.1016/S0140-6736(16)00561-4
SERGII KOLODYCH, MICHEL CHLOÉ, DELACROIX SÉBASTIEN, KONIEV OLEKSANDR, EHKIRCH ANTHONY, EBEROVA JITKA, CIANFÉRANI SARAH, RENOUX BRI: "Development and evaluation of β-galactosidase-sensitive antibody-drug conjugates", EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY, ELSEVIER, AMSTERDAM, NL, 1 August 2017 (2017-08-01), AMSTERDAM, NL , XP055416440, ISSN: 0223-5234, DOI: 10.1016/j.ejmech.2017.08.008 *
SHARPE, A.H.WHERRY, E.J.AHMED, RFREEMAN, G.J: "The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection", NAT. IMMUNOL., vol. 8, 2007, pages 239 - 245
STENTON BENJAMIN J., OLIVEIRA BRUNO L., MATOS MARIA J., SINATRA LAURA, BERNARDES GONÇALO J. L.: "A thioether-directed palladium-cleavable linker for targeted bioorthogonal drug decaging", CHEMICAL SCIENCE, ROYAL SOCIETY OF CHEMISTRY, UNITED KINGDOM, vol. 9, no. 17, 1 January 2018 (2018-01-01), United Kingdom , pages 4185 - 4189, XP093004421, ISSN: 2041-6520, DOI: 10.1039/C8SC00256H *
SUN, X. ET AL.: "Two-photon imaging of glutathione levels in intact brain indicates enhanced redox buffering in developing neurons and cells at the cerebrospinal fluid and blood-brain interface", J. BIOL. CHEM., vol. 281, 2006, pages 17420 - 17431
SURY, K.PERAZELLA, M.ASHIRALI, A.C: "Cardiorenal complications of immune checkpoint inhibitors", NAT. REV. NEPHROL., vol. 14, 2018, pages 571 - 588, XP036570646, DOI: 10.1038/s41581-018-0035-1
SUZUKI, K. ET AL.: "Glucose transporter 1-mediated vascular translocation of nanomedicines enhances accumulation and efficacy in solid tumours", J. CONTROLLED RELEASE, vol. 301, 2019, pages 28 - 41, XP085672778, DOI: 10.1016/j.jconrel.2019.02.021
THORENS, B.MUECKLER, M.: "Glucose transporters in the 21st Century", AM. J. PHYSIOL., vol. 298, 2010, pages E141 - E145, XP093045760, DOI: 10.1152/ajpendo.00712.2009
WAINWRIGHT, D.A. ET AL.: "Durable therapeutic efficacy utilizing combinatorial blockade against IDO, CTLA-4, and PD-L1 in mice with brain tumours", CLIN. CANCER RES., vol. 20, 2014, pages 5290 - 5301, XP055381858, DOI: 10.1158/1078-0432.CCR-14-0514
WANG, D. ET AL.: "Engineering nanoparticles to locally activate T cells in the tumour microenvironment", SCI. IMMUNOL., vol. 4, 2019, XP009534039, DOI: 10.1126/sciimmunol.aau6584
XIE, J. ET AL.: "Dual-sensitive nanomicelles enhancing systemic delivery of therapeutically active antibodies specifically into the brain", ACS NANO, vol. 14, 2020, pages 6729 - 6742, XP055810915, DOI: 10.1021/acsnano.9b09991
YANG TAO; MOCHIDA YUKI; LIU XUEYING; ZHOU HANG; XIE JINBING; ANRAKU YASUTAKA; KINOH HIROAKI; CABRAL HORACIO; KATAOKA KAZUNORI: "Conjugation of glucosylated polymer chains to checkpoint blockade antibodies augments their efficacy and specificity for glioblastoma", NATURE BIOMEDICAL ENGINEERING, NATURE PUBLISHING GROUP UK, LONDON, vol. 5, no. 11, 11 October 2021 (2021-10-11), London, pages 1274 - 1287, XP037617098, DOI: 10.1038/s41551-021-00803-z *
YE, Y. ET AL.: "Synergistic transcutaneous immunotherapy enhances antitumour immune responses through delivery of checkpoint inhibitors", ACS NANO, vol. 10, 2016, pages 8956 - 8963, XP055792107, DOI: 10.1021/acsnano.6b04989
ZHANG, Y. ET AL.: "A tumour-targeted immune checkpoint blocker", PROC. NATL. ACAD. SCI., vol. 116, 2019, pages 15889 - 15894, XP055736394, DOI: 10.1073/pnas.1905646116
ZHU, A. ET AL.: "Dually pH/reduction-responsive vesicles for ultrahigh-contrast fluorescence imaging and thermo-chemotherapy-synergized tumour ablation", ACS NANO, vol. 9, 2015, pages 7874 - 7885

Also Published As

Publication number Publication date
EP4342497A1 (en) 2024-03-27
JPWO2022239720A1 (en) 2022-11-17

Similar Documents

Publication Publication Date Title
CN107735090B (en) antibody-SN-38 immunoconjugates with CL2A linkers
JP2022046678A (en) Efficacy of anti-trop-2-sn-38 antibody drug conjugates for therapy of tumors relapsed/resistant to checkpoint inhibitors
ES2939760T3 (en) Cancer treatment using a chimeric receptor for antigens
CN110392570A (en) With husky trastuzumab lattice dimension for the triple negative breast cancer of health and RAD51 inhibitor for treating expression TROP-2
CN110352201A (en) The subcutaneous administration of antibody drug conjugate for cancer therapy
EP3049443A1 (en) Anti-trop-2 antibody-drug conjugates and uses thereof
Ohradanova-Repic et al. Fab antibody fragment-functionalized liposomes for specific targeting of antigen-positive cells
JP7458975B2 (en) Compositions and methods for depletion of CD5+ cells
JP7177131B2 (en) Compositions and methods for inducing apoptosis
JP2021526525A (en) Treatment method using antibody drug conjugate (ADCS)
US20210393795A1 (en) Compounds comprising cleavable linker and uses thereof
JP2018515457A (en) Calicheamicin constructs and methods of use
EP4023679A1 (en) Fusion protein targeting pd-l1 and tgf-beta and use thereof
US20220118104A1 (en) Compounds comprising cleavable linker and uses thereof
WO2022239720A1 (en) Antibody having reduced binding affinity for antigen
CN109562172B (en) Efficacy of anti-HLA-DR antibody drug conjugate IMMU-140(hL243-CL2A-SN-38) in HLA-DR positive cancers
CN110392580A (en) With topoisomerase-I inhibiting antibody-drug conjugate (ADC) treatment Small Cell Lung Cancer (SCLC) of targeting TROP-2
US20230203164A1 (en) Anti-ctla-4 monoclonal antibody, preparation method therefor, and application thereof
AU2021275489A1 (en) Single domain antibodies and their use in cancer therapies
CN114008078A (en) Bispecific antibodies against CHI3L1 and PD1 with enhanced T cell-mediated cytotoxicity on tumor cells
JP2021511333A (en) Compositions and Methods for CD134 + Cell Depletion
EP4279506A1 (en) B7-h3 chimeric antigen receptor-modified t cell and use thereof
US11879004B2 (en) Modified binding polypeptides for optimized drug conjugation
US20230210901A1 (en) Overcoming the tumor microenvironment for cell therapy by targeting myeloid derived suppressor cells through a trail-r2 specific receptor
EP4335450A1 (en) Pharmaceutical composition for prevention or treatment of lung cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22807422

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023521007

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2022807422

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022807422

Country of ref document: EP

Effective date: 20231211

ENP Entry into the national phase

Ref document number: 2022807422

Country of ref document: EP

Effective date: 20231211